Biotin causes improvements in disordered glucose metabolism by stimulating glucose-induced insulin secretion in pancreatic beta-cells and by accelerating glycolysis in liver and pancreas. Biotin is known to regulate hepatic and pancreatic glucokinase expression at both transcriptional and translational levels, and to regulate hepatic phosphoenolpyruvate carboxykinase expression at the transcriptional level. The effects of biotin on glucose-induced insulin secretion were investigated using the method of isolated pancreas perfusion. The pancreas of the biotin-deficient rat has an impaired insulin response to both glucose and arginine. In control rats as well as biotin-deficient rats, the insulin response to glucose stimulation was enhanced by the addition of 1 mM biotin to the perfusate. Biotin-induced enhancement of glucose-induced insulin release was evident within the first few minutes of perfusion. Since any effects on the glucokinase synthesis pathway would not be seen for at least 30 minutes, these results indicate that biotin may have the ability to act directly on the insulin secreting function of pancreatic beta-cells. Biotin perfusion was not found to cause enhancement of the arginine-induced insulin response, suggesting that biotin has no significant effects on the distal portion of the signaling pathway involved in insulin secretion. These results indicate that the administration of a high concentrations of biotin may improve the metabolism and/or utilization of glucose in patients with non-insulin-dependent diabetes mellitus.