How does long-term exposure to lipids inhibit glucose-induced insulin secretion? The answer to this question is central to the understanding of the impaired insulin secretion that is a hallmark of type-2 diabetes, a disorder that is characteristically associated with high plasma lipid levels. We have examined the effect of long-term (72 h) exposure of pancreatic islets to palmitate on insulin exocytosis. Pretreatment of β-cells with palmitate leads to a selective impairment of exocytosis evoked by brief (action potential-like) stimulation, whereas that evoked by long (longer than 300 ms) stimuli was unaffected. Under normal conditions, calcium influx triggered by brief membrane depolarizations increases cytoplasmic calcium within discrete microdomains and triggers the exocytosis of closely associated insulin vesicles. We found that these domains of localized calcium entry become dispersed by long-term exposure to palmitate. Importantly, the release competence of the granules was not affected by palmitate and photoliberation of caged calcium evoked indistinguishable exocytoci responses. In intact human and mouse islets, a tenfold prolongation of the beta-cell action potentials by the potassium channel blocker TEA corrected the secretion defect induced by long-term FFA exposure. We propose that the palmitate-induced dispersal of the calcium channels in beta-cells represents a mechanism to prevent insulin secretion at near-normal plasma glucose concentration when plasma FFA levels are increased during fasting/starvation.