Pseudin-2 is a cationic a-helical peptide that was first isolated from the skin of the paradoxical frog Pseudis paradoxa on the basis of its antimicrobial activity. We have investigated the insulin-releasing properties and cytotoxicity of the peptide, together with selected analogues with increased cationicity and hydrophobicity. At concentrations in the range 10(-9)-10(-6) M, pseudin-2, and its [Lys(18)], [Phe(8)], and [D-Lys(3), D-Lys(10), D-Lys(14)] derivatives, stimulated insulin release from the BRIN-BD11 clonal beta-cell line without increasing release of lactate dehydrogenase. The [Lys(18)] analogue was the most potent (46% increase in insulin release at 10(-9) M) and the most effective (215% increase in insulin release at 10(-6) M). The more cationic [Lys(3), Lys(10), Lys(14)] and [Lys(3), Lys(10), Lys(14) Lys(21)] analogues lacked insulinotropic action and the more hydrophobic [Phe(16)] analogue was cytotoxic at concentrations >= 10(-7) M. Pseudin-2 and [Lys(18)]-pseudin-2 had no effect on intracellular calcium concentrations and stimulated insulin release in the absence of external calcium. [Lys(18)]-pseudin-2 (10(-8) M) stimulated insulin release in the presence of diazoxide and verapamil. Our results demonstrate that pseudin-2 stimulates insulin secretion from BRIN-BD11 cells by a mechanism involving Ca2+-independent pathways and identify [Lys(18)]-pseudin-2 as a peptide that may have potential for development as a therapeutically valuable insulinotropic agent for the treatment of type 2 diabetes.