We studied the effects of arginine vasopressin (AVP) and oxytocin (OT) on glucagon release and characterized the receptors that mediate the effects of these two peptides by use of a number of antagonists in the perfused rat pancreas, clonal alpha-cells In-R1-G9, and fluorescence imaging of the receptors in rat islets. AVP and OT (3 pM-3 nM) increased glucagon release in a concentration-dependent manner from the rat pancreas. The antagonist with potent V1b receptor-blocking activity abolished AVP-induced glucagon release, but did not alter OT-induced glucagon release. In contrast, the OT receptor antagonist abolished OT-induced glucagon release, but did not change the effect of AVP. Fluorescent microscopy of rat pancreatic sections also showed that fluorescence-labeled vasopressin and OT bound specifically to V1b and OT receptors, respectively. Therefore, in the rat pancreas, AVP and OT increased glucagon release through the activation of V1b and OT receptors, respectively. However, in clonal alpha-cell line In-R1-G9, V1b receptors mediated both AVP- and OT-induced glucagon release, because the antagonists with V 1b blocking activity, but not the OT receptor antagonists, inhibited AVP- and OT-induced glucagon release in In-R1-G9 cells in a concentration-dependent manner. A clonal alpha-cell line In-R1-G9 was used to study the mechanisms underlying AVP-induced glucagon release. AVP (100 nM) increased [Ca 2+]i in a biphasic pattern; a peak followed by a sustained plateau. When [Ca2+]i was stringently deprived by BAPTA, a Ca2+ chelator, AVP still significantly increased glucagon release. These results suggest that AVP caused glucagon release through both Ca2+-dependent and -independent pathways. For the Ca 2+-dependent pathway, our results were consistent with the current concept that the Gq protein activates phospholipase C, which catalyzed the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 induced Ca2+ release from the endoplasmic reticulum, thereby triggering Ca2+ influx via receptor-operated Ca2+ channel and increasing glucagon release. Our results further suggest that, DAG activate novel (nPKCs) and atypical protein kinase C (aPKCs). nPKCs may exert negative feedback on AVP-induced increase in IP3 production, leading to an attenuation of [Ca2+]i, which, in turn, attenuated AVP-induced glucagon release. On the other hand, aPKCs may contribute to the stimulatory effect of AVP on glucagon release.