A low cholesterol synthetic diet containing cholesterol-4-14C of known specific activity (SA) was fed for periods of 31–45 days to 3 mongrel dogs. The SA of free cholesterol of plasma plateaued in 2 of the animals, but equilibrium was not achieved in the other. The right adrenal vein was cannulated, and, after a sufficient amount of blood had been collected to permit isolation and identification of steroids, ACTH was given and more blood was obtained. Both adrenals were removed for determination of the specific activity of free cholesterol. The specific activity of cortisol, S of Reichstein, and corticosterone was determined after the compounds had been separated and identified utilizing paper chromatography and the formation of derivatives. In all 3 animals the specific activities of the adrenal steroids, plasma free cholesterol and adrenal free cholesterol were identical. These data indicate that cholesterol is an obligatory precursor in the biosynthetic pathway of adrenocortical steroids in the dog. They also indicate that the adrenal free cholesterol either exchanges rapidly with plasma cholesterol after synthesis in situ or it is derived from the plasma cholesterol. The specific activity of plasma free cholesterol in all animals was not more than ⅓ the specific activity of the cholesterol in the diet. Thus, the majority of the circulating cholesterol in these animals had been derived by synthesis from endogenous sources. (Endocrinology74: 543, 1964)