The actions of adrenocorticotrophin 1-39 (ACTH), N6, O2-dibutyryl adenosine-3′, 5′-cyclic phosphate (dbcAMP), and human chorionic gonadotrophin (hCG) on the sulfation of DHA and the synthesis of cortisol by human fetal adrenal cells in monolayer culture have been described. Electron microscopic studies indicate that the cells present in our cultures at the end of 5 days are intact and have many of the characteristics of secretory cells. Sulfation of DHA was strongly inhibited by 11-deoxycorticosterone, testosterone, androsterone, and dihydrotestosterone. Very small effects were observed with progesterone and corticosterone and insignificant effects with estrone, estradiol, and estriol. Androstenedione as well as 17α-hydroxylated corticosteroids showed no inhibition. When used as substrate, C21 Δ4-3-ketosteroids were good precursors of cortisol with 11-deoxycortisol being the best one followed by 17α-hydroxyprogesterone and progesterone. The conversion of progesterone (4 μg/2 ml) to cortisol was fivefold greater than under conditions where no substrate was present (endogenous production). ACTH in the concentration of 5 × 10−4 U/ml added twice daily to the cell cultures for 8 days increased eightfold the sulfation of DHA and increased cortisol synthesis from progesterone 35-fold in 5 days. The addition of dbcAMP (5 × 10−5 M) or hCG used at 2.0 or 0.2 U/ml did not stimulate the sulfation of DHA. A lag period of at least 36 h is required before the stimulation of DHA sulfation or cortisol synthesis from progesterone can be observed. dbcAMP can stimulate the conversion of progesterone, 17α-hydroxyprogesterone, and 11-deoxycortisol to cortisol by the adrenal cells. These data suggest that 11β- and 21 -hydroxylation are stimulated by dbcAMP but 17α-hydroxylation is not altered.