Pancreatic β-cells possess nitric oxide (NO) synthases (NOSs) which synthesize NO and L-citrulline from L-arginine. The present study was designed to explore the mechanism of citrulline and arginine metabolism in β-cells and its possible coupling to β-cell functions. The enzymes involved in citrulline-arginine metabolism, argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and NOS were expressed in rat islets and insulinoma HIT T15 cells. In the presence of stimulatory glucose, L-citrulline and L-argininosuccinate at physiological concentrations (0.1-1 mM) increased cytosolic Ca²⁺concentration ([Ca²⁺]_i) in rat β-cells. The citrulline-induced [Ca²⁺]_iincrease was inhibited by a NOS inhibitor, N^G-monomethyl-L-arginine (NMMA). L-citrulline also stimulated NO production in HIT cells, which was inhibited by NMMA. In conclusion, L-citrulline is metabolized by ASS-ASL-NOS cycle to produce NO, which in turn increases [Ca²⁺]_iin β-cells.