The putative role of nitric oxide in the neuropathogenesis of Borna disease was investigated by determining changes in the expression of inducible nitric oxide synthase (iNOS) mRNA and constitutively expressed NOS (cNOS) mRNA in brains of Borna disease virus (BDV)-infected rats. iNOS mRNA was not detected in normal rat brain but was identified in BDV-infected brain at 14 days postinfection (p.i.), reaching maximum levels at 21 days p.i., when neurological signs and inflammatory reactions in the brain were also at a peak. cNOS mRNA was expressed in both normal brain and infected brain, increasing markedly at 17 days p.i. and reaching a peak at 21 days p.i. In situ hybridization analysis revealed iNOS mRNA in some, but not all, BDV-infected regions of the brain, particularly in the basolateral cortex and the hippocampus. iNOS-positive cells, as identified immunohistologically, were preferentially localized in perivascular areas of the hippocampus and in outer cortical layers. These iNOS-positive cells resembled monocytes/macrophages in morphology and distribution pattern but were significantly fewer. The correlation of iNOS and cNOS mRNA expression with the development of neurological disease, as well as the enhanced expression of iNOS within brain regions with inflammatory lesions, strongly suggests that NO may contribute to pathogenesis of Borna disease.