Murine polyomavirus is used in various models of persistent virus infection. This study was undertaken to assess the spatial and temporal patterns of MPyV infection in the brains of immunocompetent (BALB/c) and immunocompromised (KSN nude) mice. MPyV was stereotaxically microinfused into the brain parenchyma, and the kinetics of infection were examined by quantitative PCR. In BALB/c mice, the amount of viral DNA in the brain peaked at 4 days p.i. and then rapidly diminished. In contrast, MPyV DNA levels increased up to 4 days and then gradually decreased over the 30-day observation period in the brain of KSN mice. In both mouse strains, viral DNA was readily detected around the sites of inoculation from 2 to 6 days p.i., and continued to be detected for up to 30 days p.i. In addition, MPyV infection did not lead to a drastic induction of innate immune response in the brains, nor did MPyV-inoculated mice show any signs of disease. These results indicate that MPyV establishes an asymptomatic long-term infection in the mouse brain.