Vasopressin (VP) elicits almost identical insulin-stimulatory dose responses in isolated mouse islets and hamster β (HIT) cells. We have further pharmacologically characterized HIT cell VP receptors by comparing the potencies of a series of VP agonists including the novel V1b agonist, desamino(d-3-(3′-pyridyl)-Ala2,Arg8)VP (d(d-3-Pal)VP), in stimulating insulin secretion and inositol phosphate (IP) production. The relative orders of potency of VP analogues were parallel in both respects: desamino-Arg-VP (dAVP)>Arg-vasotocin (AVT)= VP>oxytocin (OXY)>desamino-d-Arg-VP (ddAVP)> d(d-3-Pal)VP. dAVP, the most potent agonist tested, behaved as a V1 but non-V1a agonist. The potency of d(d-3-Pal)VP relative to VP was 1:134 in stimulating insulin secretion and 1:40 with respect to IP production. In HIT cell monolayers, the relative order of affinity of analogues in competition for binding with [3 H]AVP was: dAVP>AVT=VP>V1a antagonist>OXY>ddAVP>V2 antagonist=d(d-3-Pal)VP, in parallel with their biological activity. The relative orders of potency and affinity parallel those reported for corticotrophic V1b receptors. Binding studies with hamster liver membranes indicate that the hepatic VP receptor belongs to the V1a class. We conclude that VP activates phospholipase C and interacts with functional VP receptors of the V1 type, which do not belong to the V1a subclass and which are similar to V1b receptors.