Using the highly sensitive tyramide-signal-amplification technique, we examined immunocytochemically the distribution of neuronal nitric oxide synthase (nNOS) in blood vessels of the rat brain. In contrast to the endothelial isoform, no clear-cut immunostaining could be obtained for the nNOS at the light microscopic level. An occasional faint immunoreaction at the endothelial lining was difficult to interpret. Ultrastructurally, endothelial cells of larger pial vessels, but not those of the parenchyma, revealed immunoprecipitates in their cytosol or attached to cytoplasmic membranes. Several pial and parenchymal vessels showed nNOS-positive perivascular nerves. Immunopositive and negative varicosities were located along the same fiber. The present study and our previous study reveal a clear-cut localization pattern of NOS isoforms in brain endothelium and surrounding tissue, whose particular contribution to the regulation of cerebral blood flow is still uncertain.