Species difference in the in vitro metabolic reduction of acetohexamide, an oral antidiabetic drug, was investigated using the rabbit, guinea pig, hamster, rat and mouse. The rabbit exhibited the highest acetohexamide reductase activity in cytosol of the liver and kidney among the species tested. The sensitivities to specific inhibitors of cytosolic acetohexamide reductase in the liver and kidney of the rabbit were different from those of the rat. Furthermore, species difference in acetohexamide reductase activity was found in the microsomes of the liver and kidney; only the rat and guinea pig showed significant activity.