Abstract We have recently isolated and cloned a novel endogenous peptide from pig intestine, NK-lysin (NKL). In the present study we show that NKL (1–100 nM) potently and reversibly stimulates insulin secretion in rat pancreatic islets and in the β-cell line HIT T15. This effect of NKL was not accompanied by changes in cytoplasmic free calcium concentration. The stimulatory activity of NKL on insulin release was also observed in permeabilized islets under Ca 2+-clamped conditions. Preincubation of HIT T15 cells with NKL for 1 h or 24 h did not influence cell viability. Possible mechanisms of insulinotropic activity of NKL are discussed.