The metabolism of 4-androstene-3,6,17-trione (AT), previously described as a suicide substrate for aromatase, and its irreversible binding to aromatase were studied by using human placental microsomes. AT was rapidly converted into 3 beta-reduced metabolite (3-OHAT) with an enzyme other than aromatase in the microsomes in the presence of NADPH under either aerobic or anaerobic conditions. The conversion was efficiently prevented by a steroid 5 alpha-reductase inhibitor. 3-OHAT was characterized as a competitive (Ki = 6.5 microM) and irreversible inhibitor of aromatase. Both 14C-labeled AT and 3-OHAT were demonstrated to be irreversibly bound to aromatase probably through a sulfur atom of the enzyme in time-dependent manners in the presence of NADPH, being accompanied with time-dependent losses of the enzyme activity. It was shown that the process of an apparent time-dependent loss of aromatase activity caused by AT even under conditions allowing its 3 beta-reduction should principally depend on the action of the parent inhibitor AT itself and not on that of the metabolite 3-OHAT.