Mice with deletion of insulin receptor substrate (IRS)-1 (IRS-1 knockout [KO] mice) show mild insulin resistance and defective glucose-stimulated insulin secretion and reduced insulin synthesis. To further define the role of IRS-1 in islet function, we examined the insulin secretory defect in the knockouts using freshly isolated islets and primary beta-cells. IRS-1 KO beta-cells exhibited a significantly shorter increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) than controls when briefly stimulated with glucose or glyceraldehyde and when l-arginine was used to potentiate the stimulatory effect of glucose. These changes were paralleled by a lower number of exocytotic events in the KO beta-cells in response to the same secretagogues, indicating reduced insulin secretion. Furthermore, the normal oscillations in intracellular Ca(2+) and O(2) consumption after glucose stimulation were dampened in freshly isolated KO islets. Semiquantitative RT-PCR showed a dramatically reduced islet expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b and -3 in the mutants. These data provide evidence that IRS-1 modulation of insulin secretion is associated with Ca(2+) signaling and expression of SERCA-2b and -3 genes in pancreatic islets and provides a direct link between insulin resistance and defective insulin secretion.