Acute stimulation of steroidogenesis with ACTH produces striking elevations in the plasma levels of cortisol as well as certain cortisol precursors. Although states of chronic ACTH excess are characterized by similar elevations in cortisol, the precursor levels are in the low normal range. Chronic stimulation of adrenal steroidogenesis with ACTH may enhance the activity of the late phase of cortisol biosynthesis. Precursor steroids would then be more efficiently used with less precursor leakage from the cell and greater cortisol production. In the present study, the effect of ACTH was examined on the late phase of cortisol biosynthesis in vitro using bovine adrenal cell suspensions. To exclude the well established effect of ACTH on early cortisol biosynthetic steps, the cell suspensions were treated with aminoglutethimide. This agent inhibits the conversion of cholesterol to pregnenolone and therefore to cortisol, under basal conditions and during treatment with ACTH. To aminoglutethimide-treated cell suspensions, pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone and 11-deoxycortisol were added, with and without ACTH. In the presence of ACTH, significantly more cortisol was formed from progesterone, 17-OH-progesterone and 11-deoxycortisol. To distinguish between effects of ACTH on the entry of precursors into cells and on enzyme activity within the cells, the conversion of 11-deoxycortisol to cortisol was examined in homogenized cell suspensions with and without pre-treatment with ACTH. Even in homogenized cells, prior treatment with ACTH enhanced the conversion of 11-deoxycortisol to cortisol. ACTH has a direct effect on the late phase of cortisol biosynthesis.