Abstract The production of corticosterone from 25-hydroxycholesterol by isolated rat adrenal cells is inhibited by aminoglutethimide phosphate (AGI); half-maximal inhibition is obtained at ca. 10 μM. AGI also inhibits ACTH-stimulated steroid production from endogeneous substrates; here half-maximal inhibition is obtained with ca. 40 μM AGI. In the presence of ACTH + AGI, 25-hydroxycholesterol causes additive inhibition. This effect of 25-hydroxycholesterol is dose-dependent. ACTH-stimulated steroid production from endogeneous substrates is partially inhibited by 5-cholene-3β, 24-diol. These results may just reflect substrate competition for the side-chain cleaving system or may be due to some secondary toxic effect on the cells.