We have proposed the "glucolipotoxicity" hypothesis in which elevated free fatty acids (FFA) together with hyperglycemia are synergistic in causing islet -cell damage because high glucose inhibits fat oxidation and consequently lipid detoxification. The effects of 1-2 days culture of both rat INS 832/13 cells and human islet -cells were investigated in medium containing glucose (5, 11, 20 mM) in the presence or absence of various FFA. A marked synergistic effect of elevated concentrations of glucose and saturated FFA (palmitate and stearate) on inducing -cell death by apoptosis was found in both INS 832/13 and human islet -cells. In comparison, linoleate (polyunsaturated) synergized only modestly with high glucose, whereas oleate (monounsaturated) was not toxic. Treating cells with the acyl-CoA synthase inhibitor triacsin C, or the AMP-kinase activators metformin and 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR) that redirect lipid partitioning to oxidation, curtailed glucolipotoxicity. In contrast, the fat oxidation inhibitor etomoxir, like glucose, markedly enhanced palmitate-induced cell death. The data indicate that FFA must be metabolized to LC-CoA to exert toxicity, the effect of which can be reduced by activating fatty acid oxidation. The results support the glucolipotoxicity hypothesis of -cell failure proposing that elevated FFA are particularly toxic in the context of hyperglycemia.