We previously reported that inoculation of rats with live vaccinia virus (VV) recombinants VVpyMT, VVpyLT expressing either the middle‐T (MT) or large‐T (LT) proteins of polyomavirus (PyV) can elicit immunity to challenge with syngeneic PyV‐tumor cells. We now report the results of cross‐vaccination studies. VVpyMT was ineffective against cells expressing LT protein but prevented development of MT‐expressing cells. Conversely, the VVpyLT was ineffective against MT‐expressing cells. In the two experiments performed, tumor growth enhancement rather than retardation was observed in VVpyLT‐vaccinated animals receiving PyV‐LT (FRLTI) challenge tumor cells. To determine the location of the major TSTA within MT, a further VV recombinant (VVpyMT/Cfr) was constructed that expresses only the unique C‐terminal segment of MT. VVpyMT‐Cfr and VVpyMT were equally effective in eliciting tumor immunity, indicating the presence of a major TSTA epitope within the unique C‐terminal region of MT.