The binding affinity of the differentially induced cytochrome P-450 (P-450) in rat liver microsomes with testosterone and 16α-hydroxytestosterone (16α-OH-T) was compared. Interaction of testosterone with all three microsomel P-450 of normal, MC- or PB-treated rats, gave type I spectral changes and their spectral binding constants (Ks) to P-450s were 35, 33, and 25 μM at 25℃, respectively. PB-inducible P-450s among three of these showed the highest affinity to testosterone, and the ratio of low to high spin conversion of P-450 by this compound showed also a similar tendency as the results of binding affinity. 16α-OH-T, one of testosterone metabolites, binding to all three P-450 exhibited a reversed type I spectral changes and their Ks values to normal, MC- and PB-inducible P-450 were 41, 28, and 23 μM, respectively, but binding of it with PB-inducible P-450 showed another Soret band near 460 nm. These results indicate that 16α-OH-T also binds to P-450s with a high affinity like testosterone, and it may be further metabolized to more polar compounds by P-450.