Glucose-6-phosphate dehydrogenase (G6PD) was purified from rat small intestine with 19.2% yield and had a specific activity of 53.8 units per miligram protein. The pH optimum was determined to be 8.1. The purified rat small intestinal G6PD gave one activity, one protein band on native PAGE. The observation of one band on SDS/PAGE with an M-r of 48 kDa and a specific activity lower than expected may suggest the proteolytically affected enzyme or different form of G6PD in the rat small intestine. The activation energy, activation enthalpy, Q(10), and optimum temperature from Arrhenius plot for the rat small intestinal G6Pb were found to be 8.52 kcal/mol, 7.90 kcal/mol, 1.59, and 38degreesC, respectively. The K-m values for G6P and NADP(+) were 70.1 +/- 20.8 and 23.2 +/- 7.6 muM, respectively. Double-reciprocal plots of 1/V-m versus 1/G6P (at constant [NADP(+)]) and of 1/V-m versus 1/NADP(+) at constant [G6P]) intersected at the same point on the 1/V-m axis to give V-m = 53.8 U/mg protein.