In the present study, we investigated the effects of 14 endogenous steroids on the CYP3A4-mediated drug metabolism by human liver microsomes in vitro. Nevirapine (NVP) 2-, 12-hydroxylations, carbamazepine (CBZ) 10,11-epoxidation, triazolam (TZM) 1'-, 4-hydroxylations, erythromycin (EM) N-demethylation, and 2-sulphamoylacetylphenol (SMAP) formation from zonisamide (ZNS) were investigated. The activities of the NVP 2-, 12-hydroxylations, the CBZ 10,11-epoxidation, and the TZM 4-hydroxylation were activated by endogenous androgens, such as androstenedione (AND), testosterone, and dehydroepiandrosterone. However, these androgens inhibited EM N-demethylation, TZM 1'-hydroxylation, and SMAP formation. To understand the mechanisms of these effects of androgens on CYP3A4 activities, we performed a kinetic analysis of the metabolism of CBZ and ZNS in the presence or absence of AND using the modified two-site equation model. The addition of AND to the reaction mixture caused a drastic increase in the activity of CBZ 10,11-epoxidase, especially at a low substrate concentration, and resulted in a change in the kinetics from the sigmoid to Michaelis-Menten type. On the other hand, the metabolism of ZNS was strongly inhibited by AND, although no allosteric change was observed in this case. These data demonstrate that endogenous steroids, especially androgens, strongly affect CYP3A4-mediated drug metabolism in vitro. The postulated mechanisms of the interactions between AND and CBZ or ZNS are discussed.