Neurohypophysial hormones stimulate the motility of tunica albuginea, epididymis, and vas deferens acting through oxytocin (OT) and V1 vasopressin receptors. To test the hypothesis that these hormones are involved also in the regulation of seminal vesicle physiology, we studied binding of [3H]OT and [3H] arginine vasopressin ([3H]AVP) to porcine seminal vesicle membranes. Neurohypophysial hormones bind to two different classes of sites. The first class shows low capacity (35 fmol per mg of protein) and a very high affinity (Kd less than 1 nM) for both the labeled ligands. The second class is characterized by a high capacity (2000 fmol per mg of protein) and a high affinity for AVP (Kd approximately equal to 2.5 nM), whereas OT has 160 times lower affinity. Lysine vasopressin and the V1 antagonist [1-deaminopenicillamine, 2-(O-methyl)tyrosine]Arg8-vasopressin compete with high affinity with [3H]AVP binding, whereas the V2 agonist [1-deamino,4-valine]D-Arg8-vasopressin (dVDAVP) is 110 times less potent than AVP. The OT agonist [Thr4,Gly7]OT and the OT antagonist [1(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)tyrosine, 8-ornithine]vasotocin failed to affect [3H]AVP binding. These findings seem to suggest that AVP interacts with the V1 vasopressin isoreceptor in porcine seminal vesicle membranes. However, AVP stimulates adenylate cyclase activity in a dose-dependent fashion with an EC50 of 14 nM, whereas OT or dVDAVP has no effect at 100 nM. Moreover, a well-characterized V1 vasopressin antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid),2-(O-methyl)tyrosine]Arg8-vasopressin [d(CH2)5Tyr(Me)AVP], competes with [3H]AVP binding with an IC50 of 0.17 microM. These pharmacological properties are distinct from the previously described V1 and V2 vasopressin receptors and indicate the presence of a new class of AVP receptors. Although this vasopressin isoreceptor shares some pharmacological characteristics with the V1 (pressor) isoreceptor, it has low affinity for the V1 antagonist d(CH2)5-Tyr(Me)AVP and is linked to the adenylate cyclase system. The extremely high density of AVP receptors in porcine seminal vesicles (2 pmol per mg of protein) is comparable to the density of V2 vasopressin receptors in porcine renal medulla, suggesting a physiological role for vasopressin in the seminal vesicle.