Previous work showed the existence of receptors for arginine vasopressin (AVP) in the anterior pituitary; these receptors were classified as belonging to a distinct AVP receptor subtype, referred to as AVP-V1b receptors, and are thought to mediate the well documented ACTH-releasing activity of AVP. In the present work, high affinity receptors for another neurohypophyseal hormone, oxytocin (OT), were also shown to be present within the rat anterior pituitary; to this end, [125I]d(CH2)5[Tyr(Me)2Thr4Tyr-NH2(9)]OVT was used as a ligand in receptor binding studies. Experiments on dispersed rat anterior pituitary cells in a superfusion system confirmed earlier reports that OT acts as an additional secretagogue of ACTH, with significant effects first seen at concentrations as low as 1 nM. Further studies addressed the question of whether stimulation of ACTH release is mediated by OT receptors or whether receptors for AVP (V1b receptors) might serve this role. For this, highly selective agonist and antagonist ligands of the OT receptor and nonselective agonist and antagonist ligands of the V1b receptor were employed. Neither the OT receptor agonist Thr4Gly7OT nor the OT receptor antagonist des-Gly(NH2)9d(CH2)5-[Tyr(Me)2 Thr4]OVT displayed any influence on basal ACTH release, and des-Gly(NH2)9d(CH2)5-[Tyr(Me)2Thr4]OVT did not interfere with OT-induced ACTH release; these results indicated that OT promotes ACTH release through a receptor(s) other than the OT receptor itself. Evidence for the involvement of AVP V1b receptors was provided by the observation that the AVP receptor antagonist dP[Tyr(Me2)]AVP completely abolished OT-elicited increases in ACTH release. Thus, AVP V1b receptors mediate the actions of two structurally related peptides on ACTH secretion; the role of OT receptors in adenohypophyseal function remains to be determined.