Cytomegalovirus (CMV) is the most frequent infectious cause of developmental brain disorders in humans. Here we show the role of innate immune responses caused by natural killer (NK) cells and nitric oxide (NO) derived from brain macrophages during murine CMV (MCMV) infection of the developing brain. Viral replication in the brain of newborn mice was significantly enhanced by administration of anti-asialo-GM1 antibody, specific for NK cells, or L-N6-(1-imminoethyl)-lysine, a specific inhibitor of NO synthase 2 (NOS2). These results suggest that NK cells and NO contribute to the viral clearance from the brain. At 3 days postinfection (dpi) MCMV early antigen (Ag)-positive cells were immunohistochemically detected in the periventricular area, where most of the positive cells were macrophages. At 7 dpi MCMV-Ag was found not only in cells of the periventricular area but also in neurons of the hippocampus and cortex. At 11 dpi MCMV-Ag disappeared from the periventricular area, but persisted in neurons. In the periventricular area, NK cells and NOS2-positive macrophages were associated with MCMV-Ag-positive cells. In contrast, there were very few NK cells and NOS2-positive macrophages around the MCMV-Ag-positive neurons. In situ hybridization for MCMV DNA demonstrated that positive signals were found mostly in the periventricular cells, and rarely in neurons. These results suggest that the innate immune responses are restricted to the virus-replicating cells, and do not affect MCMV-infected neurons. Therefore, evasion of the innate immune responses by MCMV-infected neurons may be an important factor in supporting the viral persistence in the developing brain.