The study investigates the impact of glucose and arginine on insulin and glucagon release in the perfused rat pancreas. Further research is required to fully comprehend how these amino acids and energy sources collectively influence pancreatic hormone secretion. We examined their effects on alpha and beta cell activity in the perfused rat pancreas, both individually and in combinations, to elucidate their mechanisms. Glucagon release is notably responsive to both glucose and arginine, underscoring distinct secretion patterns between pancreatic alpha and beta cells in response to these compounds. Varying arginine concentrations alongside 5.5 mM glucose revealed a biphasic response in insulin and glucagon release, with arginine enhancing insulin release and attenuating glucagon release compared to conditions without glucose. These findings suggest that glucose weakly inhibits arginine-induced glucagon release, influencing pancreatic alpha and beta cell dynamics through multiple pathways.