Neutralization assays were undertaken for the purpose of antigenically characterizing three strains of Tettnang virus from two geographic regions. The previously reported relationship of Tettnang virus strains to mouse hepatitis virus (MHV) was confirmed. However, the precise relationship of the Tettnang strains to prototype MHV strains was obscured in our study by the finding that the isolates had been passaged in mice from a colony subclinically infected with MHV. An Egyptian strain of Tettnang which had not been passaged in that colony was reciprocally related to the neurotropic JHM strain of MHV. Our data stress the importance of microbiological monitoring of apparently healthy laboratory animals used for virologic research.