Normal newborn rat adrenal cells kept in primary culture up to 2 1/2 months respond to ACTH stimulation and produced corticosteroid hormones and smaller amounts of 20 alpha-reduced progesterone metabolites. Cholesterol from the serum complemented culture medium serves as precursor without further addition of exogenous steroid substrates. A long-term qualitative and quantitative study of individual steroid production under the influence of ACTH was performed. ACTH treatment produced a triphasic effect on steroid production: an induction period (up to 3 days), an acute maximum production period (3rd to 6th day) and a chronic production period (to the end of the treatment). The increase in total steroid production resulted from the increase in the production of corticosteroids only. This indicated an increase of cholesterol side-chain cleavage and of the 21- and 11 beta/18-steroid hydroxylations. Removal of ACTH led to a reversible drop in total steroid production. The response to ACTH was dose dependent, so that a 2.2 mU/ml dose elicited lower steroid production than the 6.6 or 22 mU/ml doses. Increasing the lower dose after a week of treatment to a higher dose brought total steroid production and 11 beta/18-steroid hydroxylation up to the corresponding chronic production levels. The 20 alpha-steroid reduction system was not affected by ACTH. ACTH changes the importance of the two main steroidogenic pathways. With no ACTH there is approximately a 1:1 ratio between corticosteroid synthesis and progesterone reductive metabolism; a low dose of ACTH increases the total steroid production, but since corticosteroid production and 20 alpha-reduced metabolites both increase, the ratio changes little; a high dose of ACTH increases the ratio to more than 30:1. Refractoriness or desensitization to ACTH is postulated to occur through the control of cholesterol availability inside the cell possibly combined with a control of its utilization for steroidogenesis.