Utilizing a simple isotopic assay, the ability of amphenone and several of its structural analogues to inhibit the conversion of cholesterol to pregnenolone has been demonstrated in extracts of steer adrenal cortex. Inhibition by these compounds probably involves the initial enzymatic 20α-hydroxylation of cholesterol. Limited studies suggest the need for a particular spatial configuration of the 2 free amine groups of amphenone for effective inhibition. Diethylstilbestrol was also an effective inhibitor of pregnenolone biosynthesis in vitro. (Endocrinology74: 567, 1964)