Microsomal alcohol oxygenase catalyzes the stereoselective oxidation of 7alpha- and 7beta-hydroxy-delta8-tetrahydrocannabinol (7alpha- and 7beta-hydroxy-delta8-THC) to 7-oxo-delta8-THC in monkey liver, and the activity for 7beta-hydroxy-delta8-THC is relatively higher than that for 7alpha-hydroxy-delta8-THC. We previously reported that purified P450JM-E, assumed to be CYP3A8, is a major enzyme responsible for the oxidation of 7-hydroxy-delta8-THC to 7-oxo-delta8-THC in monkey liver and is capable of catalyzing the oxidative reaction by NADH as well as NADPH. In the present study, we demonstrated that some steroids such as testosterone and progesterone stimulated both the NADH- and NADPH-dependent conversions of 7beta-hydroxy-delta8-THC to 7-oxo-delta8-THC in monkey liver microsomes. Kinetic analyses revealed that both the NADH- and NADPH-dependent 7-oxo-delta8-THC formation showed sigmoid kinetics. Testosterone caused a decrease in S50 and an increase in V(max) for the NADH-dependent activity, and resulted in a decrease in S50 without changing the V(max) for the NADPH-dependent activity. On the other hand, NADH-dependent testosterone 6beta-hydroxylation activity showed Michaelis-Menten kinetics and was also inhibited by 7beta-hydroxy-delta8-THC, resulting in a decrease in V(max) with no effect on the K(m). NADPH-dependent testosterone 6beta-hydrozylation activity was also inhibited by 7beta-hydroxy-delta8-THC, resulting in a decrease in both S50 and V(max). In order to explain the metabolic interaction between 7beta-hydroxy-delta8-THC and testosterone, we propose a kinetic model involving at least three binding sites, for the mechanism of activation by testosterone.