The effects of adrenocorticotrophic hormone (ACTH) on human adrenocortical steroidogenesis were studied in adrenocortical cells which had been isolated from normal and hyperplastic glands by a technique combining tyrpsin digestion and mechanical dispersion, and incubated in the presence of ACTH or dibutyryl cyclic AMP (dbcAMP). The response was measured in terms of cyclic AMP, cortisol, corticosterone, 11-deoxycortisol and cortisone production. A classical sigmoid curve, calculated by non-linear, least square method, related the increase in cAMP production or in steroidogenesis to the log dose of ACTH. For the normal adrenocortical cells, the estimated concentration of ACTH inducing a half-maximal response was approximated 2h0 pg ACTH 1-24/ml for steroidogenesis, against 437 pg/ml for cAMP production. The estimated Vmax (per 107 cells/ml, on average) was 27 pmol cAMP/2 and for steroidogenesis (in ng/2 h): 188 for cortisol, 106 for corticosterone, 37 for 11-deoxycortisol, and 32 for cortisone, dbcAMP (1.0 mM) stimulated steroidogenesis to a comparable extent. The cells from a hyperplastic adrenal gland exhibited a steroidogenic response to ACTH and dbcAMP which was 2-3 times greater than the response of a similar number of normal adrenocortical cells. Calculated per pmol cAMP generated, the ACTH-stimulated cortisol production by cells from hyperplastic gland was also increased with respect to normal cell response. These data suggest a prolonged effect of ACTH on cortisol biosynthetic pathway beyond the membrane step of cAMP generation.