The most abundant form of estrogen circulating in fetal plasma is sulfo-conjugated estrogen; for example, estradiol-3-sulfate (E(2)SO(4)) is more highly abundant than estradiol (E(2)). The present study investigated the ontogeny of the deconjugating (steroid sulfatase [STS]) and conjugating (estrogen sulfotransferase [STF]) enzymes in ovine fetal brain and tested the hypothesis that treatment with E(2)SO(4) would alter the expression of one or both enzymes. Steroid sulfatase was more highly expressed than STF, and both changed as a function of gestational age. Estradiol-3-sulfate infused intracerebroventricularly (icv) significantly increased plasma adrenocorticotropic hormone (ACTH) and cortisol concentrations. Plasma E(2) and E(2)SO(4) were increased, and brain expression of estrogen receptor α was decreased. The proteins STS and STF were up- and downregulated, respectively. Pituitary proopiomelanocortin (POMC) and follicle-stimulating hormone (FSH) and hypothalamic corticotrophin-releasing hormone (CRH) messenger RNA (mRNA) was decreased. We conclude that E(2)SO(4) has complex actions on the fetal brain, which might involve deconjugation by STS, but that the net result of direct E(2)SO(4) icv infusion is more complex than can be accounted for by infusion of E(2) alone.