Human pancreatic islets from eight donors were incubated for 48 h in the presence of 2.0 mmol/l free fatty acid (FFA) (oleate to palmitate, 2 to 1). Insulin secretion was then assessed in response to glucose (16.7 mmol/l), arginine (20 mmol/l), and glyburide (200 micromol/l) during static incubation or by perifusion. Glucose oxidation and utilization and intra-islet triglyceride content were measured. The effect of metformin (2.4 microg/ml) was studied because it protects rat islets from lipotoxicity. Glucose-stimulated but not arginine- or glyburide-stimulated insulin release was significantly lower from FFA-exposed islets. Impairment of insulin secretion after exposure to FFAs was mainly accounted for by defective early-phase release. In control islets, increasing glucose concentration was associated with an increase in glucose utilization and oxidation. FFA incubation reduced both glucose utilization and oxidation at maximal glucose concentration. Islet triglyceride content increased significantly after FFA exposure. Addition of metformin to high-FFA media prevented impairment in glucose-mediated insulin release, decline of first-phase insulin secretion, and reduction of glucose utilization and oxidation without significantly affecting islet triglyceride accumulation. These results show that lipotoxicity in human islets is characterized by selective loss of glucose responsiveness and impaired glucose metabolism, with a clear defect in early-phase insulin release. Metformin prevents these deleterious effects, supporting a direct protective action on human beta-cells.