Polyomavirus transforms cells in culture and induces tumors in mice without apparent interaction with or inactivation of the p53 tumor suppressor protein. In this report we investigate the ability of polyomavirus T antigens to overcome the growth suppression function of p53. A temperature sensitive p53 gene was introduced into mouse embryo fibroblasts derived from a p53 null mouse, resulting in expression of a protein with a mutant conformation at 37 degrees C and a functionally wild-type conformation at 32 degrees C. We found that expression of p53 at 32 degrees C induced the cyclin-dependent kinase inhibitor p21/WAF1 and arrested cell growth in the G1/G0 phase of the cell cycle. Only the under-phosphorylated form of the retinoblastoma tumor suppressor protein (pRB) was detected in these growth arrested cells. We introduced both polyomavirus large T (LT) and middle T (MT) antigens into this cell line and showed that LT overcame p53-dependent growth arrest, while MT did not. In cells grown at 32 degrees C, LT expession led to cell proliferation and phosphorylation of pRB in the presence of p21. A mutant LT containing a defective pRB binding domain failed to overcome the growth arrest, indicating that interaction of LT with RB proteins is required to override p53 function. Although the polyomavirus T antigens do not interact with p53 directly, our results indicate that the virus, through LT, is able to interfere with the growth suppressive activity of p53.