Glibenclamide, a "second generation" sulfonylurea, produced the same pattern of insulin release from the perfused rat pancreas as did tolbutamide. The stimulatory effect was closely dependent on the glucose concentration present. Both agents enhanced insulin secretion at 5--10 mM glucose, whereas no additional insulin was released when maximally stimulating levels of glucose (20 and 30 mM) were present. The concentrations of glibenclamide stimulating insulin release were 100--400 times lower than equieffective levels of tolbutamide. At glucose levels of 3 or 8 mM, however, glibenclamide did not liberate significantly more insulin from the pancreas than did tolbutamide. Thus the differences of tolbutamide and glibenclamide were quantitative rather than qualitative. Although the active concentrations differed the effects produced were comparable.