Sulphonylureas stimulate insulin secretion by binding to a receptor in the pancreatic beta-cell plasma membrane resulting in inhibition of ATP-sensitive K+ channels, membrane depolarization and thus influx of Ca2+ through voltage-dependent Ca2+ channels. Sulphonylureas can also induce hormone release at fixed membrane potentials without Ca2+ entry suggesting that these drugs may have other modes of action. We have determined whether different forms of sulphonylurea-binding proteins are present in insulin-secreting cells and their subcellular localization by density gradient centrifugation. Binding studies using [3H]-glibenclamide showed that islet and insulinoma membranes contained a single high affinity sulphonylurea binding site (Kd = 1 nmol/l). Photo-crosslinking of the drug to the membranes resulted in labelling of two proteins with apparent molecular weights of 170 and 140 kDa. The same analyses of insulinoma subcellular fractions showed that the majority (> 90%) of binding proteins were localized to intracellular membranes with only minor levels (< 10%) on plasma membranes. The 170 kDa sulphonylurea binding protein was present in both plasma and granule membrane fractions whereas the 140 kDa form was not present in the plasma membrane fraction. The differences in the molecular forms and subcellular distribution of the receptor are consistent with sulphonylureas having multiple sites of action in the pancreatic beta cell.