The inhibitory effect of an anabolic steroid, stanozolol, on testicular microsomal cytochrome P-450 (17α-hydroxylase/C_{17, 20}-lyase)(P-450_17α/lyase) and the nature of the interaction were compared with those of other anabolic steroids, furazabol and mestanolone. Stanozolol markedly inhibited Δ¹⁶-C₁₉-steroid synthesizing activity, 17α-hydroxylase and C_{17, 20}-lyase activities, which were mediated by oxygenase activities of testicular microsomal cytochrome P-450_17α/lyase. In addition, stanozolol was a competitive inhibitor of 17α-hydroxylase (K_i=6.31 μM) and C_{17, 20}-lyase (K_i=1.30 μM) activities in the reconstituted enzyme system.The interaction of cytochrome P-450_17α/lyase with stanozolol induced a type I difference spectrum (peak at 387 nm and trough at 418 nm) with a dissociation constant (K_s) of 1.47 μM.