To examine the prevalence of aflatoxins in poultry feeds and to substantiate the effects of aflatoxin B1 on broilers’ performance, a study was conducted on commercial poultry feeds and their ingredients during 2006- 2007. A total of 216 samples comprised of wheat, maize, rice, cotton seed meal, broiler starter and finisher rations were collected from local poultry markets of Peshawar, Swat and D. I. Khan regions of North West Frontier Province (NWFP) of Pakistan. Sampling was carried out during the winter (December-February), spring (March-May), summer (June-August) and autumn (September-November) seasons of the year 2006/2007. It was found that water activity (aw) of samples collected from Peshawar, Swat and D.I. Khan regions ranged from 0.500 to 0.834 aw, 0.408 to 0.815 aw, and 0.554 to 0.747 aw, respectively. Seasonal variation significantly affected water activity of the samples. Moisture sorption isotherms of the samples showed that all the commodities were hygroscopic in nature. The dominant fungal genera isolated from the feeds were Penicillium spp (25.45 %), Aspergillus spp (19.01%), Rhizophus spp (14.32%), Mucor spp (10.52%), Fusarium spp (10.69%), and Eurotium spp (9.46%) across all the three regions. Total fungal viable count ranged from 6.45 to 26.69 x 103, 6.80 to 16.90 x 103 and 6.43 to 25.58 x 103 CFUs g-1 in samples from Peshawar, Swat and D. I. Khan regions, respectively. It was observed that total fungal count significantly varied among the substrates and different seasons. Total culturalable A. flavus and A. parasiticus in the samples from Peshawar ranged from 1.51 to 21.42 x 102 CFUs g-1 and 2.27 to 32.14 x 102 CFUs g-1, while that in Swat and D. I. Khan samples ranged from 1.51 to 42.52 x 102 CFUs g-1, 2.27 to 63.79 x 102 CFUs g-1 and 1.57 to 102.30 x 102 and 2.36 to 153.45 x 102 CFUs g-1, respectively. It was observed that 63.33% of all the isolates of Aspergillus sec Flavi were aflatoxigenic in nature while the rest were non-aflatoxigenic. All commodities were found contaminated with aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2), the ranges being 13.71 to 191.65, 5.46 to 86.85, 8.55 to 167.82 and 5.14 to 89.90ng g-1, respectively in the samples from Peshawar region. Samples from Swat region had AFB1, AFB2, AFG1 and AFG2 in the range of 13.37 to 147.34, 3.63 to 22.38, 4.27 to143.33, and 1.95 to 49.72 ng g-1 whereas that of D. I. Khan region contained in the range of 6.96 to 58.31, 3.39 to 27.92, 4.41 to 24.54 and 0.02-4.46 ng g-1, respectively. The main enzymes produced by germinating conidia of A. flavus were esterase, lipase, acid phosphatase, β-glucosidase and N-acetyl-β-D- glucosaminidase, while for A. parasiticus these were alkaline phosphatase, lipase, acid phosphatase and β-fucosidase in terms of both total (μmol 4- nitrophenol min-1 g-1) and specific activity (nmol 4-nitrophenol min-1 μg-1 protein). There were significant increases in the specific activity of all these enzymes of germinating spores of A. flavus and A. parasiticus for up to 72 hrs. The total/specific activities of the enzymes produced by A. flavus were maximum at 0.99 aw, with the exception of acid phosphatase and N-acetyl-β- D-glucosaminidase at 0.94 aw. For A. parasiticus, maximum total activity occurred at 0.99 aw but specific activity was found to be higher at lower aw levels. Calcium propionate, aw and incubation time, alone and interactively, significantly affected total fungal viable count and aflatoxins production in both starter and finisher broiler rations. Minimum culturalable fungi were counted in calcium propionate added feeds at lower aw (0.85aw) level at the start of experiment which progressively increased over time, increasing aw levels and decreasing concentration of calcium propionate in the feeds. Similar trends were observed for aflatoxins (B1, B2, G1 and G2) production in both starter and finisher broiler rations. Study in vitro on A. flavus (A-2092) and A. parasiticus (PRR-2747) showed that conidia of both species were germinated on all calcium propionate (0.5 and 1%) and aws (0.996, 0.96 and 0.94 aw) treatments, however, 1% calcium propionate at 0.94aw delayed the germination process for up to 10 and 9 days in A. flavus and A. parasiticus, respectively. The growing rates of spores of both species were slower at 1% calcium propionate and 0.94 aw. Aflatoxins (B1, B2, G1 and G2) were also minimally produced by A. flavus and A. parasiticus at 1% calcium propionate dose and 0.94 aw. However, none of the treatments completely inhibited the growth and aflatoxins production by A. flavus and A. parasiticus. Broilers’ feed intake, body weight gain, feed conversion ratio, meat of carcass and dressing percentages were significantly affected by consumption of AFB1 contaminated feed. Feed intake, average body weight gain and meat of carcass were significantly reduced with increasing levels of AFB1 in the feed. On the contrary, feed conversion ratio and dressing percentages were increased as the level of aflatoxins increased in the feed. Residues of AFB1 and AFM1 were detected in both liver and muscles of chicks only when they were fed with higher level (>20ng g-1) of AFB1 in the feed. Comparatively, higher residual levels of aflatoxins B1 and M1 were observed in the liver than in the muscles showing that liver is the primary site of metabolism of aflatoxins in chicken.