6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, Delta H, Q(10) and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4 degrees C, respectively. The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with K-m values of 595 +/- 213 mu M for 6PG and 53.03 +/- 1.99 mu M for NADP. 1/V-m versus 1/6PG and 1/NADP plots gave a V-m value of 8.91 +/- 1.92 U/mg protein. NADPH is the competitive inhibitor with a K-i of 31.91 +/- 1.31 mu M. The relatively small K-i for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.