This study examined the effects of various drugs and sterols on the rate of 7alpha-hydroxycholesterol synthesis in isolated rat liver microsomes. Cholesterol 7alpha-hydroxylase activity was significantly inhibited by proadifen (98%), metyrapone (67%), and aminoglutethimide (45%) at concentrations of 1 mM and by ascorbic acid (40%) at a concentration of 10 mM. Cimetidine had no significant effect. The activity of cholesterol 7alpha-hydroxylase was also significantly inhibited by 7beta-hydroxycholesterol (38%) and 7-ketocholesterol (35%) at concentrations of 1 micro M, and by 10 micro M 7alpha-hydroxycholesterol (35%), 25alpha-hydroxycholesterol (32%), and 5-cholenic acid-3beta-ol (27%). Two bile acids, cholate and lithocholate, as well as a geometric isomer of cholesterol, coprostanol, had little influence on 7alpha-hydroxylase activity at concentrations of 10 micro M. The inhibitory effect of metyrapone was additive with that of either 7beta-hydroxycholesterol or proadifen; the effects of 7beta-hydroxycholesterol and proadifen were not additive. These results suggest that proadifen and 7beta-hydroxycholesterol interact with the same enzyme site while metyrapone binds at a different location. Proadifen inhibited 7alpha-hydroxylase irreversibly, while kinetic studies demonstrated noncompetitive inhibition by metyrapone (K(I) = 0.55 mM) and competitive inhibition by 7beta-hydroxycholesterol (K(I) = 2.4 micro M). The inhibition of 7alpha-hydroxylase activity by metyrapone and aminoglutethimide, drugs used to manage patients with excessive cortisol production, suggests that such treatment may also alter bile acid synthesis.-Schwartz, M. A., and S. Margolis. Effects of drugs and sterols on cholesterol 7alpha-hydroxylase activity in rat liver microsomes.