PPAR alpha is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. PPAR alpha is involved in the regulation of in vivo triglyceride levels, presumably through its effects on fatty acid and lipoprotein metabolism. Some nuclear receptors have been involved in rapid effects mediated by non-genomic mechanisms. In this paper, we report the rapid non-genomic effects of PPAR alpha ligands on the intracellular calcium concentration ([Ca2+]i), mitochondrial function, reactive oxygen species (ROS) generation, and secretion of insulin in freshly isolated mouse islets of Langerhans. The hypolipidemic fibrate PPAR alpha agonist WY-14 643 decreased the glucose-induced calcium oscillations in intact islets. This effect was mimicked by the synthetic agonist GW7647 and the endogenous agonist oleylethanolamide. The WY-14 643 action was rapid in onset (5 min) and was still produced in the presence of protein and mRNA synthesis inhibitors, cycloheximide, and actinomycin-d. This suggests that it is independent of gene transcription. In addition, WY-14 623 impaired mitochondrial function, increased ROS formation and decreased insulin release. PPAR alpha is present in beta-cells, mainly in the cytosol and nucleus, with a small subpopulation localized in the plasma membrane. However, the presence of the PPAR alpha ligand effects in mice bearing a disrupted Ppar alpha gene raises the possibility that the rapid effects of the agonists in pancreatic beta-cells are independent of the receptor. We conclude that PPAR alpha agonists produce a decrease in glucose-induced [Ca2+]i signals and insulin secretion in beta-cells through a rapid, non-genomic mechanism.