Corticotrophs in the fetal sheep become increasingly responsive to arginine vasopressin (AVP) in late gestation. We previously reported that this may be due in part to corresponding increases in signal transduction (inositol 1,4,5-trisphosphate, IP(3)). These ontogenic changes are prevented by hypothalamo-pituitary disconnection (HPD), which also prevents fetal plasma cortisol concentrations from increasing in late gestation. This led us to hypothesize that cortisol is involved in mediating the changes in pituitary responsiveness. HPD was performed on fetal sheep at 120 days gestational age (dGA). Half of the HPD fetuses were infused with cortisol for 3 days beginning at 135-137 dGA (HPD+C). The remaining HPD fetuses and a group of sham-operated control fetuses were infused with saline. Pituitary cells were isolated and cultured. After 48 h, a subset of cells was stimulated with 100 nM AVP for 2 h, and the medium was collected for ACTH analysis. Another subset of cells was stimulated with 100 nM AVP for 30 min, and the formation of IP(3) was determined. Plasma cortisol concentrations increased rapidly within the first 6 h after infusion (5.2 +/- 1.9 to 29.7 +/- 4.9 ng/ml) but did not increase thereafter. Cells from HPD+C and sham-operated fetuses secreted significantly more ACTH than those from HPD fetuses (% increase from control: 33.0 +/- 8.8%, 47.9 +/- 10.6%, and 11.9 +/- 2.4%, respectively). IP(3) formation was significantly increased in cells from HPD+C and sham-operated compared with HPD fetuses (% increase from control: 17.7 +/- 4.4%, 18.9 +/- 4.3%, and 4.6 +/- 1.5%, respectively). These findings support the idea that cortisol plays a role in mediating the increase in pituitary responsiveness to AVP in the late-gestation fetal sheep.