Chlorpyrifos an insecticide, is extensively used in Pakistan and contaminating the soil and water environments at several sites. Exploration of efficient chlorpyrifos-degrading bacteria to clean-up this toxicant is of immense importance. This study reports the isolation, screening and identification of highly efficient chlorpyrifos degrading bacterial strains from different soils and water samples collected from different sites of Pakistan. Out of total 50 isolated bacterial strains, 32 were tested for their biodegradation capability in a culture medium containing chlorpyrifos as carbon and energy source. The bacterial isolates showed a great variation (25-92%) in their ability to degrade chlorpyrifos in broth condition. Out of these 32 strains, four bacterial strains (SWLC2, SWLH2, SGB2 and SWLC1) were the most promising in their growth and biodegradation activity and were identified and characterized by biochemical characteristics and 16S rRNA sequence analyses. Three strains (SGB2, SWLC1 and SWLC2) showed the greatest similarity to the members of Enterobacteriacea and one strain (SWLH2) to Rhizobiacea families. These selected strains (Enterobacter sp. SWLC1, Enterobacter sp. SGB2, Agrobacterium sp. SWLH2 and Enterobacter sp. SWLC2) exhibited biodegradation potential between 81 to 92% of the spiked amount of chlorpyrifos (100 mg l -1 ) within 18 days of incubation in broth culture. Biodegradation of chlorpyrifos continued gradually throughout the incubation period (18 days) as examined by the HPLC - UV system. Abiotic degradation contributed only up to 15% of the spiked amount. Maximum biodegradation by the four efficient bacterial strains was observed at an initial pH of 7 and an incubation temperature of 30 o C, under shaking conditions. Among these four strains, Enterobacter sp. SWLC2 was found most efficient in biodegradation of chlorpyrifos and was selected for further studies. This strain was then optimized in broth as well as in soil slurry under different conditions. Enterobacter sp. SWLC2 showed maximum biodegradation of chlorpyrifos at pH 7, 30 o C, under shaking conditions with inoculum size of 800 μl. Biodegradation of chlorpyrifos by Enterobacter sp. SWLC2 varied in different textured soils, being more rapid in course textured soils than in fine textured soil. Among these soils, loamy soil was selected for further studies in soil slurry experiments due to its medium textured nature as soils of Punjab are mostly loamy in texture. An initial inoculum size of 800 μl (OD= 0.80), incubation temperature of 30 o C, under shaking, initial pH 7 of soil slurry of loam soil were found optimal conditions for maximum biodegradation of chlorpyrifos byEnterobacter sp. SWLC2. Exogenous application of sugars, yeast extract, manitol, organic acids and amino acids had stimulator or inhibitory effects on biodegradation of chlorpyrifos by Enterobacter sp. SWLC2. Among these, glucose, yeast extract, succinic acid and citric acid had stimulatory effects on biodegradation of chlorpyrifos. Biodegradation of chlorpyrifos by Enterobacter sp. SWLC2 was also checked at different initial concentrations of chlorpyrifos from 10-250 mg l -1 . Rate of biodegradation increased with increase in concentration of chlorpyrifos from 100-150 mg l -1 and suggested first order rate kinetics. In all experiments, utilization of chlorpyrifos by the strains was accompanied by a parallel increase in optical densities of broth implying that removal of this pesticide from the growth medium was a growth linked biodegradation. These results highlighted the potential of this bacterium to be used in the detoxification strategies of chlorpyrifos contaminated water and soil environments.