SUMMARY: Adrenocortical cells obtained from adult rats were propagated in monolayer culture. Depending on culture conditions, they grew either as lipid-containing epithelial-like cells with a high level of steroid production, or as fibroblast-like cells with a low level of steroid production. The major fluorogenic steroid secreted by both morphologic forms of adrenal cortical cell was corticosterone as determined by chromatography and acid fluorometry. Basal fluorogenic steroid production per 106 cells over 24 h was: epithelial-like cells, 5·0 μg; fibroblast-like cells, 0·014 μg. Stimulation with ACTH for 5 days increased fluorogenic steroid production and induced morphologic changes in both adrenal cell forms. ACTH stimulation of fluorogenic steroid production by both cell forms reached a maximum after 3 days, then dropped to a refractory state after 5 days. With maximal ACTH stimulation, production increased 25-fold in fibroblast-like cells and five-fold in epithelial-like cells. The latter rate of corticosterone production is similar, per cell, to ACTH-stimulated adrenal glands in vivo. Progressive morphologic changes were observed with ACTH stimulation: epithelial-like cells retracted from the substratum and lost lipid inclusions; fibroblast-like cells became more epithelial-like. Both adrenal cell types formed intermediates from [4-14C]pregnenolone including pregn-5-ene-3β,20α-diol and 20α-hydroxypregn-4-en-3-one. Control cultures of muscle fascia fibroblasts did not produce corticosterone or intermediates from [4-14C]pregnenolone and did not respond to ACTH functionally or morphologically.