Different hormones of the hypothalamo-pituitary-adrenal axis (corticotropin-releasing factor, adrenocorticotropic hormone, corticosterone) were measured in brain pieces (stalk, median eminence, hypothalamus), hypophyses, adrenals and plasma of 21-day-old rat fetuses from mothers which were given either plain tap water or water containing dexamethasone acetate (10 micrograms/ml) from day 15 to 21 of gestation. Dexamethasone induced drastic reduction of body weight (-66% vs. controls), severe atrophy of the adrenals (-83%) and a sharp drop in their corticosterone content (-74%). Fetal plasma corticosterone levels were below the lower limit of detection of the competitive corticosteroid-binding globulin (CBG) radioassay (less than 0.01 microgram/ml). Both atrophy and severe reduction of the adrenal activity in fetuses from dexamethasone-treated females were in good correlation with a drastic decrease in plasma adrenocorticotropic hormone (ACTH) levels which were below the lower limit of detection of the radioimmunoassay (RIA) used (less than 10 pg/ml) and a significant reduction in pituitary ACTH content (-93%). The low corticostimulating activity of the fetal hypophyses was associated with a drop in both corticotropin-releasing factor (CRF) hypothalamic content (-57%) and concentration (-67%). The effects of dexamethasone on plasma and pituitary ACTH concentrations in 21-day-old fetuses were compared to those, previously reported, of encephalectomy and decapitation performed on day 16 of gestation. The reported data were consistent with the present results, suggesting both pituitary and hypothalamic sites for the in vivo inhibiting action of dexamethasone on the rat hypothalamic-pituitary-adrenal axis in late gestation.