The effects of tolbutamide on insulin, glucagon, and somatostatin secretion were investigated in the isolated perfused pancreas from normal and diabetic rats under low (30 mg/dl), normal (100 mg/dl), and high (300 mg/dl) glucose conditions. In the normal rat pancreas, tolbutamide-induced insulin release was increased when the glucose concentration in the perfusion medium was increased from 30-300 mg/dl. Tolbutamide had an inhibitory effect on glucagon release at the low (30 mg/dl) glucose concentrations, although a stimulatory effect was observed under normoglycemic conditions. The total amount of somatostatin secretion above baseline during tolbutamide infusion was higher under the normal glucose than under the low glucose condition. However, further augmentation of somatostatin release was not found at the high glucose concentration. In the diabetic rat pancreas, insulin release was diminished and tolbutamide-induced somatostatin release was enhanced with increasing glucose concentrations. Glucagon release was stimulated at the normal glucose concentration, but inhibited temporarily at the high glucose concentration. The maximum somatostatin response in the early phase was significantly decreased in the diabetic pancreas under low and normal glycemic conditions, when expressed as an incremental change (percentage) above baseline. From these results, one can conclude: (1) tolbutamide has a stimulatory effect on the pancreatic D cell in both the normal and diabetic pancreas; (2) the early response of somatostatin is decreased in the diabetic pancreas, except under conditions of high glucose concentration; and (3) the pancreatic A cell response to tolbutamide was not uniform and was quite different from the response of the D cell.