The mechanism by which long-term exposure of the β-cell to elevated concentrations of fatty acid alters glucose-induced insulin secretion has been examined. Exposure of INS-1 β-cells to 0.4 mmol/l oleate for 72 h increased basal insulin secretion and decreased insulin release in response to high glucose, but not in response to agents acting at the level of the K_ATP channel (tolbutamide) or beyond (elevated KCl). This also suppressed the glucose-induced increase in the cellular ATP-to-ADP ratio. The depolarization of the plasma membrane promoted by glucose was decreased after oleate exposure, whereas the response to KCl was unchanged. Cells exposed to free fatty acids displayed a lower mitochondrial membrane potential and a decreased glucose-induced hyperpolarization. The possible implication of uncoupling protein (UCP)-2 in the altered secretory response was examined by measuring UCP2 gene expression after chronic exposure of the cells to fatty acids. UCP2 mRNA and protein were increased twofold by oleate. Palmitate and the nonoxidizable fatty acid bromopalmitate had similar effects on UCP2 mRNA, suggesting that UCP2 gene induction by fatty acids does not require their metabolism. The data are compatible with a role of UCP2 and partial mitochondrial uncoupling in the decreased secretory response to glucose observed after chronic exposure of the β-cell to elevated fatty acids, and suggest that the expression and/or activity of the protein may modulate insulin secretion in response to glucose.