Vasopressin (AVP) receptors present in In-R1-G9 cells, a hamster glucagon-secreting alpha-pancreatic cell line, were characterized using SSR-149415, a selective nonpeptide V1b receptor antagonist, and reference AVP compounds. Binding experiments, using [3H]AVP as a ligand, identified a single population of high-affinity binding sites. SSR-149415 competitively inhibited this binding and exhibited nanomolar and stereospecific affinity for these sites. The affinity of various AVP/oxytocin ligands confirmed a V1b binding profile. In functional studies, AVP was a potent stimulant in inducing intracellular Ca2+ increase, glucagon secretion, and cell proliferation. These effects were fully antagonized by SSR-149415 with a nanomolar potency, whereas its diasteroisomer as well as two selective V1a and V2 receptor antagonists were much less potent. Additionally, the order of potency of AVP agonists and antagonists was in agreement with V1b-mediated effects. By RT-PCR, we confirmed the presence of V1b receptor mRNA in both In-R1-G9 cells and in human pancreas. The distribution pattern of V1b receptors investigated in human pancreas by immunohistochemistry showed strong labeling in islets of Langerhans, and colocalization studies indicated that this receptor was expressed in alpha-glucagon, beta-insulin, and somatostatin pancreatic cells. Thus, in In-R1-G9 cells, AVP mediates intracellular Ca2+ increase, glucagon secretion, and cell proliferation by activating V1b receptors, and these effects are potently antagonized by SSR-149415. Moreover, the presence of V1b receptors also found in human Langerhans islets could suggest hormonal control of AVP in human pancreas.