We studied the actions of tolbutamide on the release of somatostatin, insulin, and glucagon from the isolated, perfused dog pancreas. Tolbutamide (180 μm) elicited a biphasic response of all three islet hormones in the absence of glucose. In the presence of normal glucose (5.5 mm), 180 μm tolbutamide was again stimulatory, however, now the D cell response appeared uniphasic and the relative increase of somatostatin was lower than in the absence of glucose. At the highest perfusate glucose of 11 mm, no augmentation of the somatostatin output was seen to 180 μm tolbutamide, whereas the B and A cell secretion was still stimulated. In dose-response studies with tolbutamide (1.8–1800 μm) it was found that the D cell threshold to tolbutamide was 18 μm, Furthermore, that maximal D cell secretion was attained in the presence of 180 μm tolbutamide at low glucose (1.4 mm) and of 18 μm tolbutamide at normal glucose (5.5 mm), respectively. The insulin and glucagon responses showed clearcut dose-dependency over the range of tolbutamide doses applied. The B and A cell threshold to tolbutamide was 1.8 μm when the prevailing glucose level was stimulatory for the B and A cell, respectively. The finding that D and B cell responses to tolbutamide were eliminated during calcium deprivation indicates a key role of calcium in the events that proceeds to tolbutamide-mediated somatostatin and insulin release. In conclusion, the primary effect of tolbutamide on the islet cells is to stimulate hormone secretion, however, pertubations in terms of appearance and magnitude of D, B, and A cell responses depend on the balance between the concentrations of tolbutamide and glucose.