Polyoma virus is highly oncogenic when inoculated into immunocompromised adult mice and neonatal mice of specific inbred strains. Although T lymphocytes are known to be essential in controlling polyoma virus tumorigenesis, the importance of class I MHC-restricted CD8+ T cells in mediating tumor resistance remains unclear. Here, we investigated the tumorigenicity of polyoma virus in adult mice rendered CD8+ T cell-deficient by homozygous (-/-) disruption of the beta 2-microglobulin (beta 2m) or CD8 alpha (CD8) genes. Nearly all (94%) of the virus-infected adult C57BL/6 beta 2m-/- mice developed tumors, and 20% of the virus-inoculated adult C57BL/6CD8-/- mice developed hindlimb paralysis, which is indicative of vertebral tumors. Only 2 of 20 virus-inoculated adult normal C57BL/6 mice developed tumors. Despite these different tumor susceptibilities, persistent viral DNA was detected in multiple organs of mice of all three strains. Multifocal lymphoplasmacytic interstitial infiltrates were present in the kidneys and lungs of virus-infected C57BL/6 beta 2m-/- and in the lungs of virus-inoculated C57BL/6CD8-/- mice. These infiltrates were composed primarily of B cells and colocalized with staining for the major viral capsid protein, VP1. No infiltrates or VP1 staining was detected in the kidneys of infected C57BL/6 mice. Using a highly sensitive RT-PCR bioluminescence immunoassay, we investigated and detected persistent polyoma T protein and VP1 messages in both C57BL/6 beta 2m-/- and C57BL/6 mice. C57BL/6 beta 2m-/- and C57BL/6 mice had equivalent serum virus-neutralizing antibody titers. These results provide in vivo evidence that class I MHC-restricted CD8+ T cells are involved in mediating protection against polyoma virus tumor development.