The present study revealed that glucose-induced insulin secretion was significantly inhibited by perfusion with trypsin (10 mug/ml) in the isolated rat pancreas perfusion. However, when trypsin was interposed during perfusion of 16.6 mM glucose, glucose-induced insulin secretion was not suppressed during trypsin infusion. The insulin level was significantly lower in the trypsin treated animals than in the control after completion of trypsin infusion. The results suggest that trypsin does not attack the glucorecoptor bound with glucose but attacks the free glucoreceptor. Interestingly a biphasic pattern of glucose-induced insulin secretion disappeared by the pretreatment with trypsin. The possibility was suggested that tyrpsin prevented the initial binding of glucose to the B-cell.