Interactions between glucose and amino acids in rat pancreatic islets were studied by recording the intracellular membrane potential and spike discharges from single islet cells and by measuring insulin release from the isolated perfused pancreas. It was found that L-isoleucine requires the presence of basal glucose (5 mM) in Order to increase spike discharge from islet cells and depolarize the cell membrane. Similarly basal glucose is needed for insulin release by L-isoleucine. A physiological mixture of twenty amino acids also required the presence of basal glucose in order to increase spike activity and insulin release. In contrast to L-isoleucine the amino acid mixture did not depolarize the β-cells. Iodoacetate, at concentrations previously shown to block glycolysis completely, did not interfere with any of these permissive actions of glucose, nor did iodoacetate alter the well known electrical manifestations of high levels of glucose itself (i.e. depolarization and increased spike discharge). These data show that glucose plays a pre-eminent role as regulator of islet cell function, governing the efficacy of amino acids as β-cells stimulants. The results are most easily interpreted if one assumes that glycolysis is not required for glucose to exert its action.