Theiler's murine encephalomyelitis virus (TMEV), a member of the genus Cardiovirus, is an enteric pathogen of mice that causes acute encephalomyelitis followed by persistent central nervous system infection with chronic inflammation and demyelination after intracerebral inoculation. Although TMEV is a mouse pathogen, antibodies against TMEV strain GDVII have been detected in conventional rat colonies. Natural infection of rats by Cardiovirus has not yet been described. The purpose of this study was to demonstrate TMEV infection of rat colonies by using serologic assays, reverse transcription-polymerase chain reaction (RT-PCR) analysis, and clinical characterization. Indirect immunofluorescence assay of rat serum samples demonstrated antibodies against TMEV-GDVII in 86.3% of samples analyzed, and 77.2% of the antibody-positive samples had neutralizing antibodies. To determine whether rats can be infected experimentally with TMEV-GDVII, specific pathogen-free newborn mice and rats were inoculated intracerebrally with intestinal suspensions from seropositive rats. Both species showed the typical clinical signs of TMEV infection in mice, which is characterized by flaccid hindlimb paralysis and tremor. RT-PCR in brain tissue of experimentally infected animals detected RNA sequences corresponding to the 5' noncoding region of Cardiovirus known as the 'internal ribosome entry site.' These results suggest that rats can be naturally infected with TMEV and related Cardiovirus. Therefore, continued health monitoring for TMEV infection should be included in rat colonies mainly because these animals are used for various experimental purposes.