Glycolysis and gluconeogenesis are highly conserved metabolic pathways in all the three domains of life. These pathways are slightly modified and contain some unusual enzymes including bifunctional enzymes. In these pathways phosphofructokinase step is the rate-limiting step. Fructose 1, 6-bisphosphatase and phosphofructokinase catalyze the opposite reactions at this step. Another important step is the sixth step of glycolysis where two molecules of glyceraldehydes 3-phospjate enter the second half of glycolysis producing 1, 3- biphosphoglycerate catalyzed by glyceraldehyde 3-phosphate dehydrogenase. I chose these two steps and analyzed the enzymes involved at these steps in hyperthermophilic archaeon Pyrobaculum calidifontis. The present study reports on cloning and characterization of fructose 1,6-bisphosphatase aldolase (Pcal_0111), Phosphofructokinase domain containing protein (Pcal_0041) and glyceraldehyde 3- phosphate dehydrogenase (Pcal_0632) from P. calidifontis. In the genome sequence of P. calidifontis Pcal_0111 is annotated as fructose bisphosphate aldolase displaying high highest homology of 92% with uncharacterized fructose bisphosphate aldolase from Pyrobaculum arsenaticum. Among the characterized enzymes Pcal_0111 exhibited highest identity of 91% with the enzyme from Thermoproteus neutrophilus. Purified recombinant Pcal_0111 catalyzed both phosphatase and aldolase reactions with specific activity values of 4µmol-1min-1mg-1 and 1.3µmol-1min-1mg-1 respectively. These values are highest among the fructose 1, 6-bisphosphatases/aldolases characterized from archaea. The enzyme activity increased linearly with the increase in temperature till 100 °C. Recombinant Pcal_0111 was highly stable with a half-life of 120 min in the boiling water. The enzyme activity was not affected by AMP but strongly inhibited by ATP with an IC50 value of 0.75 mM. Pcal_0111 exhibited wide substrate specificity in contrast to previously characterized fructose 1, 6- bisphosphatases/aldolases. Genome sequence of P. calidifontis contains an open reading frame, Pcal_0041, annotated as phosphofructokiase B domain containing protein/possible phosphofructokinase. Pcal_0041 exhibited highest identity of 78% with uncharacterized ribokinase from Pyrobaculum islandicum. Among the characterized enzymes, it displayed a 37% identity with PFKB from Aeropyrum pernix. Heterologous expression of the gene resulted in production of recombinant protein was in the soluble form. Purified recombinant Pcal_0041 exhibited a low phsphofructokinase activity but high phosphofructokinase and pyrimidine kinase activities. There was continous increase in activity with increase in temperature until 90 oC with activation energy of 67kJ/mol. Pcal_0041 is the most thermostable ribokinase characterized to date with half-life of 90 min in boiling water. It was active at wide pH range and displayed almost equivalent amount of the activity at pH 7‒10. The genome sequence of P. calidifontis also contained an open reading frame, Pcal_0632, annotated as glyceraldehyde 3-phosphate dehydrogenase. Pcal_0632 exhibited highest homology of 83% with the corresponding uncharacterized enzymes from P. oguniensis and P. islandicum. Among characterized enzymes it displayed a 69% identity with the enzyme from Thermoproteus tenax. Recombinant Pcal_0632 was produced in inclusion bodies. Gene expression was optimized by using different induction temperatures. Nearly 50% soluble production of the protein was obtained at 17 oC overnight induction after a heat shock of 20 min at 45 oC. Pcal_0632 could utilize both NAD and NADP as cofactors but 2-fold higher activity with NADP. Optimal pH and temperature for the enzyme activity were 7.0 and 85 oC. Pcal_0632 was highly thermostable with a half-life of 26 h at 85 oC. There was a 5 fold increase in activity in the presence of MgCl2. In conclusion the three glycolytic/gluconeogenic enzymes from P. calidifontis are novel and highly thermostable. High thermostability and inhibition by ATP make Pcal_0111 a unique fructose 1, 6-bisphosphatase/aldolase. High specific activity with ribose 1-phosphate, cytidine and uridine indicates that Pcal_0041 might be a bona-fide ribose 1-phosphate kinase and pyrimidine kinase that probably plays a dual role in ribose and pyrimidine metabolism.