Thirty-four brains with microglial nodular brain stem encephalitis were retrospectively investigated for herpes simplex virus (HSV) and cytomegalovirus (CMV) by in situ hybridization (ISH) with biotinylated cDNA probes, and by immunocytochemistry with polyclonal and monoclonal antibodies on formalin fixed paraffin embedded serial tissue sections. In 16 cases (47%), HSV DNA was found by ISH in the nuclei of neurons in microglial nodules or in the adjacent parenchyma of the brainstem, and more rarely at various cerebellar and telencephalic sites. None of the 34 cases was labeled for CMV DNA and none revealed HSV or CMV antigens. Ten control brains without microglial nodules were not labeled. This study suggests an HSV etiology for many cases with microglial nodular brainstem encephalitis.