Corticotrophs in the fetal sheep become increasingly responsive to arginine vasopressin (AVP) in late gestation. We have previously reported that this may be due in part to corresponding increases in signal transduction (inositol trisphosphate, IP3). 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 of gestation (dGA). Half of the HPD fetuses were infused with cortisol for 3 days beginning on 135-137 dGA (HPD+C). The remaining HPD fetuses and a group of sham operated controls were infused with saline. Pituitary cells were isolated and cultured. After 48h, a subset of cells was stimulated with 100nM AVP for 2h, and the medium collected for ACTH analysis. Another subset of cells was stimulated with 100nM AVP for 30mins and the formation of IP3 determined. Plasma cortisol concentrations increased rapidly within the first 6h following infusion (5.2±1.9 to 29.7±4.9 ng/ml), but did not increase thereafter. Cells from HPD+C and sham fetuses secreted significantly more ACTH than those from HPD fetuses (% increases from control were 33.0±8.8, 47.9±10.6 and 11.9±2.4). IP₃ formation was significantly increased in cells from HPD+C and sham compared to HPD fetuses (% increases from control were 17.7±4.4, 18.9±4.3 and 4.6±1.5). 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.