The inhibitory effects (IC50) of 16-methyl steroids on 5 alpha-reductase were studied. The in vitro experiments were carried out with homogenates of rat and human prostates. The investigated 16-methyl steroids were found to be weak inhibitors. In comparison with the known 5 alpha-reductase inhibitor 4-MA, the relative IC50 values of the studied compounds are 4.7 times or more greater than 4-MA in human prostate and 23.5 times or more greater than 4-MA in rat prostate. The IC50 values increase in the sequence 16 alpha, 16-beta- and 16,16-dimethyl derivatives. In human prostate homogenates IC50 varies between 0.6 and 120, while in rat it ranges from 1.6 to 1000 microM. This shows that the enzyme of the human prostate is more sensitive than that of the rat prostate to the methyl-substituted compounds. Acylation of the 17-hydroxy group significantly increases the IC50 values (cf. 8,11: from 4.8 to 23.5 and from 0.52 to 0.62;9,12: from 26.0 to 170.0 and from 0.58 to 1.4;15,17: from 3.9 to 35.0; and 16,18: from 5.6 to 58.0 microM). Whereas lack of a 19-CH3 group improves the inhibitory effect in the 16-unsubstituted compounds (1,19;14,22), the reverse hold in the 16-methylated derivatives (9,20; 10,21; 15,23; 16,24).