A technique of monolayer tissue culture of human fetal adrenal cells was developed in order to study steroidogenic responses to factors such as ACTH. The daily production of 12 steroids [pregnenolone, 17-hydroxy pregnenolone, dehydroepiandrosterone (DHA), DHA sulfate, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, corticosterone, 11-desoxycortisol, cortisol, and aldosterone) was measured by RIA. Initially, fresh fetal adrenal cells produced DHA, DHA sulfate, 17-hydroxypregnenolone, and small amounts of cortisol, but in the absence of ACTH, the production of all steroids declined during culture to low levels. The addition of physiological amounts (1-10(4) pg/ml) of either alpha ACTH-1(1-24) or alpha ACTH-(1-39) or coculture with fetal pituitary cells elicited a progressive rise in steroid production during the first 4-6 days of incubation. The lowest ACTH doses elicited a proportionately greater adrenal androgen response (as reflected in the DHA to cortisol ratio), but with increasing ACTH dosage, there was greater stimulation of cortisol production, which equalled or exceeded that of DHA. The data demonstrate that fetal adrenal cells may be maintained in short term culture and can respond to physiological amounts of ACTH. The progressive increase in the production of cortisol and other delta 4, 3-ketosteroids in vitro suggests that the characteristic fetal pattern of steroidogenesis may result from the interaction of ACTH with some circulating inhibitor of adrenal 3 beta-hydroxysteroid dehydrogenase.