The glycosphingolipid sulfatide is present in secretory granules and at the surface of pancreatic beta-cells, and antisulfatide antibodies (ASA; IgG1) are found in serum from the majority of patients with newly diagnosed type 1 diabetes. Here we demonstrate that sulfatide produced a glucose- and concentration-dependent inhibition of insulin release from isolated rat pancreatic islets. This inhibition of insulin secretion was due to activation of ATP-sensitive K(+)-(K(ATP)) channels in single rat beta-cells. No effect of sulfatide was observed on whole-cell Ca(2+)-channel activity or glucose-induced elevation of cytoplasmic Ca(2+) concentration. It is interesting that sulfatide stimulated Ca(2+)-dependent exocytosis determined by capacitance measurements and depolarized-induced insulin secretion from islets exposed to diazoxide and high external KCl. The monoclonal sulfatide antibody Sulph I as well as ASA-positive serum reduced glucose-induced insulin secretion by inhibition of Ca(2+)-dependent exocytosis. Our data suggest that sulfatide is important for the control of glucose-induced insulin secretion and that both an increase and a decrease in the sulfatide content have an impact on the secretory capacity of the individual beta-cells.