Aromatase and 5 beta-reductase activity are expressed at high levels in the zebra finch brain, especially in the telencephalon. Aromatization of androgens to estrogens is thought to be a critical step in the organization and activation of avian sexual behaviors. In contrast, 5 beta-reductase is thought to be an inactivating enzyme, one that catalyzes the conversion of androgens to biologically inactive metabolites. To address the importance of aromatase activity in this system, it is necessary to find an effective and selective aromatase inhibitor, one that has little or no effect on other androgen-metabolizing enzymes. The potency and specificity of fadrozole hydrochloride as an aromatase inhibitor was tested in zebra finch telencephalon. The compound was tested in vitro in primary dissociated cell cultures made from hatching telencephalon and compared to a commonly used inhibitor, 1,4,6-androstatriene-3,17-dione (ATD). Untreated, these cultures express extremely high levels of aromatase and 5 beta-reductase activity and therefore allow sensitive measurement of the effectiveness of inhibitors. Aromatase activity was also measured in homogenates of adult telencephalon following in vivo fadrozole injection. Finally, aromatase and 5 beta-reductase activity were quantified in zebra finch telencephalon following similar intramuscular injections in 4- to 6-day-old birds. In all three cases, fadrozole was highly effective in reducing aromatase activity. Fadrozole increased 5 beta-reductase activity, presumably due to an increase in available substrate, but had no inhibitory effect on the enzyme. ATD was less effective in inhibiting aromatase, and it also inhibited 5 beta-reductase activity at high concentrations.