Alyteserin-2a (ILGKLLSTAAGLLSNL.NH2) stimulated the rate of insulin release from BRIN-BD11 clonalβ cells at a concentration of 30 nm (p < 0.05) with a response of 296 ± 26% of basal release at 3 μm (p < 0.001). The insulinotropic actions of analogs containing substitutions by l-lysine, d-lysine, or l-tryptophan at sites that maintain amphipathicity were evaluated. The [G11K], [S7k], [S7k,G11k], and [G11k,N15K] analogs were the most potent stimulating insulin release at 0.01 nm (p < 0.05). The [S7K], [G11K], [S14K], [N15K], [G11k], and [S7K,G11K] analogs were the most effective producing an approximately twofold greater (p < 0.001) release of insulin at 3 μm compared with alyteserin-2a. The [T8W] and [A9W] analogs were less active than alyteserin-2a. No peptide-stimulated release of lactate dehydrogenase at concentrations up to 3 μm, indicating that the integrity of the plasma membrane had been preserved. Membrane depolarization and an increase in intracellular Ca2+ concentration are involved in the mechanism of action of the peptides. Administration of [G11k]alyteserin-2a (75 nmol/kg body weight) to high-fat-fed mice with obesity and insulin resistance significantly (p < 0.01) enhanced insulin release and improved glucose tolerance during the 60-min period following an intraperitoneal glucose load.