In sheep, the fetal hypothalamus-pituitary-adrenal axis plays a central role in the initiation of parturition. We have reported that estradiol dramatically increases the activity of the fetal hypothalamus-pituitary-adrenal (HPA) axis. Sulfoconjugated estrogens are known to circulate in high concentrations in fetal plasma. We have reported the expression and abundant activity of steroid sulfatase within the fetal brain regions important for HPA axis control, and we have proposed that sulfoconjugated estrogens in fetal plasma are deconjugated (and therefore converted to a biologically active form) in fetal brain. The present study was designed to test the hypothesis that exogenous estradiol-3-sulfate stimulates HPA axis activity in late gestation fetal sheep and that it is concentrated by fetal brain tissue. We infused estradiol-3-sulfate iv into fetal sheep (125-135 d gestation; term = 147 d) at rates of 0, 0.25, and 1.0 mg/d for 5 d and performed serial sampling of fetal blood before and at the end of the infusion periods. Infusions increased fetal plasma estradiol-3-sulfate concentrations and produced dose-related increases in HPA axis activity. The action of the steroid on the fetal brain was also demonstrated as dose-related increases in the abundance of Fos in fetal cerebellum. In a second study we measured the uptake of sulfoconjugated and unconjugated estrogen (estrone-3sulfate and estrone, respectively) into the fetal brain (124-128 d gestation) in vivo. Both forms of estrogen were concentrated in fetal brain, with the uptake of estrone greater than that of estrone-3-sulfate. We conclude that sulfoconjugated estrogens augment fetal HPA axis activity and that they can cross the fetal blood-brain barrier. We propose that in late gestation the large circulating pool of sulfoconjugated estrogen is a biologically important source of active hormone that might play a role in the timing of parturition in sheep.