Free fatty acids (FFA) cause a rise in cytosolic free Ca2+ ([Ca2+]i) and stimulate insulin release from pancreatic beta-cells. The G-protein coupled receptor GPR40 can be activated by medium- and long-chain FFA. We investigated a potential role for GPR40 in the generation of the FFA-induced Ca2+ signal and insulin secretion. [Ca2+]i was measured in primary mouse beta-cells and in INS-1 cells, and insulin secretion was assessed from INS-1 cells. GPR40 expression was determined by RT-PCR and downregulation of GPR40 expression by siRNA transfection was carried out in INS-1 cells. A number of saturated, mono- and polyunsaturated medium- and long-chain FFA caused a rise in [Ca2+]i both in primary mouse beta-cells and in INS-1 cells. By contrast, the short-chain saturated caproic acid was ineffective at concentrations up to 300 microM. In INS-1 cells, the FFA-induced Ca2+ signal required mobilization of internal Ca2+ and Ca2+ influx through voltage-sensitive Ca2+ channels. RT-PCR analysis revealed that GPR40 is expressed in INS-1 cells. Downregulation of GPR40 by specific siRNA treatment lead to a significant inhibition of the FFA-induced [Ca2+]i response and insulin secretion, indicating that the FFA-stimulated Ca2+ signal and insulin secretion involve activation of GPR40 in pancreatic beta-cells.