We have examined the localization of inducible nitric oxide synthase (iNOS) and nitrotyrosine (the product of nitration of tyrosine by peroxynitrite, a highly reactive derivative of nitric oxide [NO]) in demyelinating lesions from (i) two young adult patients with acute multiple sclerosis (MS), (ii) a child with MS (consistent with diffuse sclerosis), and (iii) five adult patients with chronic MS. Previous reports have suggested a possible correlation between iNOS, peroxynitrite, related nitrogen-derived oxidants, and the demyelinating processes in MS. We have demonstrated iNOS-immunoreactive cells in both acute-MS and diffuse-sclerosis-type lesions. In acute-MS lesions, iNOS was localized in both monocytes/macrophages and reactive astrocytes. However, foamy (myelin-laden) macrophages and the majority of reactive astrocytes were iNOS negative. In specimens from the childhood MS patient, iNOS protein was present only in a subpopulation of reactive or hypertrophic astrocytes. In contrast, no iNOS staining was detected in chronic-MS lesions. Immunohistochemical staining of acute-MS lesions with an antibody to nitrotyrosine revealed codistribution of iNOS- and nitrotyrosine-positive cells, although nitrotyrosine staining was more widespread in cells of the monocyte/macrophage lineage. In diffuse-sclerosis-type lesions, nitrotyrosine staining was present in hypertrophic astrocytes, whereas it was absent in chronic-MS lesions. These results suggest that NO and nitrogen-derived oxidants may play a role in the initiation of demyelination in acute-MS lesions but not in the later phase of the disease.