The production of large amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been described as a double-edged sword eliciting pro- or anti-inflammatory effects in different immune situations. Our aim, therefore, was to investigate its role in experimental autoimmune uveoretinitis (EAU), a model of ocular inflammation, induced in the Lewis rat following a single footpad injection of retinal Ags. iNOS enzyme was not detected in the normal Lewis rat eye, but was strongly expressed by infiltrating ED1+ macrophages during the acute inflammatory stages of EAU. Treating immunized animals with L-arginine increased urinary NO metabolite (NOx) levels, accelerated the inflammatory response, and increased disease severity, whereas treatment with the NOS inhibitor, N(G)-nitro-L-arginine methyl ester, reduced NOx excretion, delayed the onset, and reduced the clinical signs of EAU. Reverse transcription-PCR analysis of ocular tissue from untreated and treated animals detected iNOS mRNA at all stages of disease, and expression was up-regulated during peak disease. L-arginine treatment enhanced cytokine mRNA expression, particularly of IFN-gamma, which was detected earlier than in control animals, corresponding with the more rapid onset of disease and increased disease severity observed in this group. N(G)-nitro-L-arginine methyl ester had little or no effect on iNOS or inflammatory cytokine mRNA expression. These results suggest NO is central to the pathogenesis of EAU and highlight the importance of the macrophage as an effector cell in what is considered a CD4+ T cell-dependent disease. Furthermore, this study demonstrates the therapeutic potential of NOS inhibitors in the treatment of inflammatory and autoimmune mediated disease.