Hyperoxaluric kidneys show impaired diuretic response to acute infusion of L-arginine. In this study, we studied the effects of chronic L-arginine supplementation on CaOx crystal formation in hyperoxaluric rat kidneys. Eight groups were tested: the control group received drinking water, L group received L-arginine (0.6%), LN group received N^G-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), L+LN group received L-arginine plus L-NAME, HP group received hydroxyl-L-proline (HP, 5%) mixed with chow to induce hyperoxaluria, L+HP group received HP plus L-arginine, HP+LN group, and L+HP+LN group. The duration was 42 days and each group had n=8. Urinary biochemistry and renal CaOx amounts were measured, as well as renal expressions of NOS isoforms and NAD(P)H oxidase. The distribution of iNOS, NAD(P)H oxidase, ED1-positive cells, and nitrotyrosine were examined by immuno-histochemical and immuno-fluorescence studies, while superoxide production from the kidneys was examined by fluorescence spectrometric assay. Compared to the HP group, the L+HP group had excessive CaOx crystal accumulation and enhanced eNOS, iNOS, and NAD(P)H oxidase protein expression in the kidney. Urinary excretion of nitrotyrosine was markedly increased. Increased superoxide formation in the L+HP kidney was derived from NAD(P)H oxidase and uncoupled eNOS, and increased nitrotyrosine formation might derive from iNOS and ED1-positive cells which gathered around the CaOx crystals. L-NAME co-treatment (L+HP+LN group) reduced renal oxidative, nitrosative stress, and tubular damage which were induced by L+HP. The results showed that chronic L-arginine treatment to the hyperoxaluric kidney with many CaOx crystal deposition may have a toxic effect by enhancing intra-renal oxidative and nitrosative stress.