Pregnenolone accumulates in rat adrenal sections incubated in the presence of cyanoketone, and it is thus possible to directly study the net conversion of cholesterol to pregnenolone. ACTH and cyclic-AMP stimulate net pregnenolone synthesis to a degree which is sufficient to fully explain their effects on corticosterone production. Calcium and continued protein synthesis are directly required for the ACTHinduced stimulation of pregnenolone synthesis. Despite accumulation of pregnenolone within the adrenal tissue during incubation in media without added protein, there is no apparent inhibition of pregnenolone synthesis. The latter suggests that cholesterol side-chain cleavage is not significantly affected by the accumulated pregnenolone (Endocrinology89: 958, 1971)