Dehydroepiandrosterone sulfate is the major steroid secretory product of the human fetal adrenal gland. Several factors have been shown to modulate the secretion of this steroid by cultured fetal adrenal cells. In addition to the cytochrome P450 enzymes that are important in steroid biosynthesis, dehydroepiandrosterone sulfotransferase (DST) is likely to be a key regulated enzyme in the formation of sulfated steroids, which are characteristic of the human adrenal cortex, particularly that of the fetus and the adult zona reticularis. In the present investigation, we sought to evaluate the cellular localization of DST in cultures derived from the fetal zone, neocortex, and adrenal capsule and to determine the effects of ACTH and other agonists of the protein kinase-A pathway on the abundance of DST in such cells. Cells derived from the fetal zone, neocortex, and adrenal capsule were either precultured for 3-13 days in plastic flasks followed by culture on coverslips or were cultured directly on coverslips in control medium (McCoy's 5A medium that contained 5% fetal bovine serum) or control medium plus ACTH, forskolin, or dibutyryl cAMP for 1-4 days. Cells were fixed in buffered formalin and then immunostained for DST by use of a rabbit polyclonal antiserum prepared against human liver DST. DST immunoreactivity was abundant in freshly isolated cortical cells derived from fetal zone and neocortex. DST immunoreactivity was still observable in fetal zone and neocortex cells as well as in cells prepared from enzymatic digests of adrenal capsule after scraping off adherent neocortex cells following culture for 9-14 days in control medium. Adrenal fibroblasts were negative for DST. DST abundance in cortical cells was increased in cultures supplemented with ACTH, forskolin, or dibutyryl cAMP compared to that in cultures grown in control medium alone. The results of Western blot analyses of DST in these cells were consistent with the immunocytochemical data. These results suggest that DST is present in both fetal zone and neocortex cells of the human fetal adrenal at midgestation and that the production of DST is stimulated by ACTH and agonists of the protein kinase-A signal transduction pathway in the human fetal adrenal gland. © 1995 by The Endocrine Society.