Arginine vasopressin (VP) activates phospholipase C (PLC) and stimulates insulin secretion and inositol phosphate (insP) production in mouse islets and clonal hamster beta cells (HIT). The insulin response to VP is reportedly dependent on extracellular glucose and there is evidence that glucose also activates PLC. The interactions of VP and glucose have been further examined in HIT cells. Glucose stimulated insulin secretion but not insP production and VP stimulated both insulin secretion and insP production in the absence of extracellular glucose. However, in the presence of glucose the insulin and insP responses to VP were potentiated. The phorbol ester, tetradecanoylphorbol acetate (TPA), which activates protein kinase C (PKC), stimulated basal insulin secretion but inhibited insP production. In the presence of submaximal concentrations of VP, 100 nmol/l TPA inhibited VP-stimulated inositol monophosphate production and there was no additivity of stimulated insulin secretion. With a maximal (10 nmol/l) concentration of VP, TPA caused additive insulin secretion and insP levels were stimulated above baseline. Twenty-four hour preincubation with TPA to down-regulate PKC did not inhibit the insulin response to VP. We conclude that extracellular glucose does not activate PLC and is not required for VP-stimulated insulin secretion although it potentiates VP-stimulated insulin secretion and insP production.