The uptake of 3H-labelled glibomuride wasstudied in microdissected pancreatic islets of ob/ob-mice. The islets rapidly accumulated this insulin-releasing sulfonylurea derivative in amounts exceeding the sucrose space. Steady-state was reached within 5 min, suggesting that the drug was bound to the β-cell plasma membrane. The uptake of glibomuride was greater than that of tolbutamide but smaller than that of glibenclamide. It was significantly inhibited by serum albumin and, to a lesser extent, by glibenclamide. Tolbutamide as well as four analogues of tolbutamide and glibenclamide that lack blood-sugar-lowering capacity had no significant effect on the uptake of glibenclamide. It is suggested that the insulin-releasing potency of sulfonylureas depends on the magnitude of their binding to the β-cell plasma membrane.