The enzyme, 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) is an essential element in the biosynthetic pathway for potent adrenal steroid hormones that appear to regulate maturation of many tissues in utero and are critical for homeostasis after birth. The results of prior studies are suggestive that 3 beta-HSD activity in the human fetal adrenal (HFA) is very low and restricted to the outer zone of cortical cells, the neocortex (NC), during mid-gestation. Near the time of birth, however, there must be enhanced expression of this enzyme to allow for adaptation to extrauterine life. In the present study, we sought to characterize, by use of immunohistochemical methods, the cellular localization and developmental changes of 3 beta-HSD in the HFA during the interval of 11-41 wks gestation. Early in gestation, 11-15 wks, we noted considerable 3 beta-HSD in NC and in occasional fetal zone (FZ) cells as well. Thereafter until 24-25 wks, 3 beta-HSD was very low in NC cells and virtually absent from the FZ. Throughout the third trimester, the outer 1/2-2/3 of the NC was increasingly immunostained and clusters of immunoreactive cells also appeared near the central medullary vein of the adrenal. The NC cells and those located in the cortical cuff region that expressed 3 beta-HSD resembled zona glomerulosa cells. Among many other fetal tissues studied, only testicular Leydig cells (18,19 wks) and hilar cells of the ovary (26 wks) were found to contain 3 beta-HSD in quantities sufficient to be detected by immunohistochemistry. These results are suggestive of a heretofore undocumented stimulus to 3 beta-HSD in the HFA in early gestation followed by a suppression of the adrenal concentration of this enzyme during mid-gestation. High levels of 3 beta-HSD in early development may facilitate cortisol production, which is believed to play a role in differentiation of the medullary precursors during this developmental period. The control of adrenal 3 beta-HSD during human fetal development may be more complex than initially envisioned and requires further study.