1. The role of L-arginine in the basal and stimulated generation of nitric oxide (NO) for endothelium-dependent relaxation was studied by use of NG-monomethyl L-arginine (L-NMMA), a specific inhibitor of this pathway. 2. L-Arginine (10-100 microM), but not D-arginine (100 microM), induced small but significant endothelium-dependent relaxations of rings of rabbit aorta. In contrast, L-NMMA (1-300 microM) produced small, endothelium-dependent contractions, while its enantiomer NG-monomethyl-D-arginine (D-NMMA; 100 microM) had no effect. 3. L-NMMA (1-300 microM) inhibited endothelium-dependent relaxations induced by acetylcholine (ACh), the calcium ionophore A23187, substance P or L-arginine without affecting the endothelium-independent relaxations induced by glyceryl trinitrate or sodium nitroprusside. 4. The inhibition of endothelium-dependent relaxation by L-NMMA (30 microM) was reversed by L-arginine (3-300 microM) but not by D-arginine (300 microM) or a number of close analogues (100 microM). 5. The release of NO induced by ACh from perfused segments of rabbit aorta was also inhibited by L-NMMA (3-300 microM), but not by D-NMMA (100 microM) and this effect of L-NMMA was reversed by L-arginine (3-300 microM). 6. These results support the proposal that L-arginine is the physiological precursor for the basal and stimulated generation of NO for endothelium-dependent relaxation.