In late gestation fetal sheep the pituitary becomes increasingly responsive to stimulation by arginine vasopressin (AVP). This change appears to be one important factor mediating the plasma cortisol surge, a critical developmental event. It is not known precisely why pituitary corticotropes become more responsive at this time. In this study we examined the possibility that changes in second messenger generation (inositol trisphosphate, IP₃) are responsible. Two studies were undertaken. The first was an ontogeny study where pituitaries were isolated from 100, 120, and 140 days gestational age (dGA) fetal sheep. Cells were cultured, stimulated with arginine vasopressin (AVP) and the formation of IP₃ assessed. The amount of IP₃ generated increased with gestational age (% increases from unstimulated controls were 4.6, 11.5 and 21.5 for 100, 120 and 140 dGA respectively), with significant differences between the 140 dGA group and both earlier groups apparent. The second study examined the impact of 120 dGA hypothalamo-pituitary disconnection (HPD- which prevents corticotrope maturation) on responsiveness of pituitary cells isolated from 140 dGA fetuses. Cells were stimulated with AVP, and the formation of IP₃ and secretion of ACTH were assessed. Significantly less IP₃ was formed and ACTH secreted in cells from HPD compared with control fetuses (IP₃ and ACTH levels were 50% and 35% lower respectively). Results from the HPD study demonstrate that the ontogenic changes in IP₃ following AVP require an intact hypothalamic-pituitary adrenal axis. These findings suggest that heightened second messenger generation may be a key reason for increased ACTH secretory responsiveness to AVP in the late gestation sheep fetus.