The role of nitric oxide (NO) in the pathogenesis of viral encephalitis was investigated by using an experimental model of herpes simplex virus type 1 (HSV-1) encephalitis in Lewis rats. The expression of inducible NO synthase (iNOS) mRNA determined by Northern blotting was observed first in the olfactory bulb and the brain stem on day 5 after intranasal inoculation of HSV-1, and thereafter iNOS mRNA was detected in other brain regions, i.e., cerebrum and cerebellum. In various parts of the brain, excessive NO production was identified by electron spin resonance spectroscopy. The temporal and spatial patterns of iNOS expression coincided with those of viral propagation, as demonstrated by polymerase chain reaction for HSV-1 gene expression as well as by the plaque-forming assay. Immunohistochemical study determined that iNOS was localized mainly in monocyte-derived macrophages. Treatment of virus-infected animals with the NOS inhibitor N(ω)-monomethyl-L-arginine (L-NMMA), but not N(ω)-monomethyl-D-arginine, significantly ameliorated not only clinical symptoms such as paralysis and seizures but also mortality. Virus yield from brain tissue was not affected by L-NMMA treatment. It is of interest that increased expression of the antioxidant enzyme heme oxygenase-1 was observed in the HSV-1 -infected brain; this increased expression was strongly inhibited by L-NMMA treatment. These data suggest that the high level of NO produced by iNOS is a pathogenic factor in HSV-l-induced encephalitis in rats.