Adult bovine adrenal cortical cells in monolayer culture were used to study the induction of cholesterol side-chain-cleavage cytochrome P-450 by corticotropin (ACTH). In the presence of 1 microM ACTH, there was a 4-fold increase in cortisol production by these cells over a 72-hr period and a corresponding increase in total cytochrome P-450 content. The incorporation of [35S]methionine into a number of cellular proteins was stimulated by the presence of ACTH in the culture medium, whereas the incorporation into other proteins was decreased. The temporal profile of these changes varied from one protein to another. Examination of the incorporation of [35S]methionine into mitochondrial protein showed an increased production of a radiolabeled protein that comigrated with the form of cytochrome P-450 known as side-chain-cleavage cytochrome upon incubation with ACTH. Thus, it appears that the cytochrome P-450scc content is increased in bovine adrenal cortical cells exposed to ACTH. Cytochrome P-450scc, synthesized in a cell-free translation system directed by RNA isolated from bovine adrenal cortical tissue or from cells, had a molecular weight of 54,500. Cytochrome P-450scc isolated from bovine adrenal mitochondria had a molecular weight of 49,000. Thus, cytochrome P-450scc is synthesized as a larger precursor that must be processed by proteolytic cleavage before or upon insertion into the mitochondrion.