1. Cytochromes P-450sec and P-450(11) beta, adrenodoxin and adrenodoxin reductase, mitochondrial components of the adrenocortical steroid hydroxylase pathway, are synthesized as higher molecular weight precursors; cytochrome P-450C-21, a microsomal component of this pathway, is synthesized as the mature form. 2. Synthesis of the above mitochondrial components is induced by ACTH in a co-ordinated fashion. Synthesis of cytochrome P-450C-21 and NADPH-cytochrome P-450 reductase is also induced by ACTH, however, the induction of these microsomal components is not co-ordinated with that of the mitochondrial components. 3. Following treatment of cultured cells with ACTH, the pattern of glucocorticoid output changes from approximately equal amounts of cortisol and corticosterone to predominately cortisol within 24 h. This change results from a large induction of cytochrome P-450(17) alpha activity in response to ACTH. 4. Bovine adrenocortical cells in culture become refractory to continued treatment with ACTH. This refractoriness is manifested in terms of steroid output; synthesis of cytochromes P-450scc, P-450(11) beta and P-450C-21, adrenodoxin and adrenodoxin reductase; and activities of cholesterol side-chain cleavage, 11 beta-hydroxylase and 17 alpha-hydroxylase.