Human fetal adrenal glands obtained at mid-gestation were incubated in vitro, and steroids in the incubation media and tissues were measured by radioimmunoassay. During two consecutive one-hour incubation periods, mean concentrations of 2.23+/-0.45 and 2.77+/-0.60 ng/h/mg tissue (+/-SE) of combined pregnenolone and pregnenolone-sulfate were found in the media of the control group. When adrenocorticotropin (ACTH) was added in the second half of incubation, the total pregnenolone level rose from an average of 1.90+/-0.42 to 5.89+/-2.00 ng/h/mg tissue (p less than 0.05). The dehydroepiandrosterone (DHA) and DHA-sulfate levels in the control media remained almost unchanged, while the addition of ACTH caused a significant increase, from 6.26+/-1.48 to 15.49+/-4.08 ng/h/mg tissue (p less than 0.05). The tissue content of these steroids increased slightly but not significantly in the ACTH group. Lesser amounts of 16 alpha-hydroxy-DHA and 16 alpha-hydroxy-DHA-sulfate, progesterone and cortisol were also measured in both incubation media and tissue. Concentrations of these steroids did not change significantly upon the addition of ACTH in vitro. This in vitro study further supports the hypothesis that the fetal pituitary-adrenal axis has the potential to respond to stress-induced ACTH release and secretes steroids which are the precursors of sex steroids in pregnancy.