1. We have investigated the mechanism by which L‐arginine stimulates membrane depolarization, an increase of intracellular calcium ([Ca2+]i) and insulin secretion in pancreatic beta‐cells. 2. L‐Arginine failed to affect beta‐cell metabolism, as monitored by NAD(P)H autofluorescence. 3. L‐Arginine produced a dose‐dependent increase in [Ca2+]i, which was dependent on membrane depolarization and extracellular calcium. 4. The cationic amino acids L‐ornithine, L‐lysine, L‐homoarginine (which is not metabolized) and NG‐monomethyl‐L‐arginine (L‐NMMA, a nitric oxide synthase inhibitor) produced [Ca2+]i responses similar to that produced by L‐arginine. The neutral nitric oxide synthase inhibitors NG‐nitro‐L‐arginine (L‐NNA) and N omega‐monomethyl‐L‐arginine (L‐NAME) also increased [Ca2+]i. D‐Arginine was ineffective. 5. L‐Arginine did not affect whole‐cell Ca2+ currents or ATP‐sensitive K+ currents, but produced an inward current that was carried by the amino acid. 6. The reverse transcriptase‐polymerase chain reaction demonstrated the presence of messenger RNA for the murine cationic amino acid transporters mCAT2A and mCAT2B within the beta‐cell. 7. L‐Arginine did not affect beta‐cell exocytosis as assayed by changes in cell capacitance. 8. Our data suggest that L‐arginine elevates [Ca2+]i and stimulates insulin secretion as a consequence of its electrogenic transport into the beta‐cell. This uptake is mediated by the mCAT2A transporter.