Polyomavirus productively infects mouse cells, transforms rat fibroblasts in culture, and induces a broad spectrum of tumors when inoculated into newborn mice. The expression of large, middle, and small T antigen are necessary for virus growth and oncogenic transformation. We have generated a small deletion in a region common to both large and middle T antigen that encodes three consecutive prolines. In this report we characterize this mutant virus in terms of its ability to replicate in mouse cells, transform rat fibroblasts, and induce tumors in the mouse. We find that the virus immortalizes primary cells and that viral DNA replication is not impaired, indicating that these functions of large T antigen are not altered. However, the ability of the virus to transform rat fibroblasts is defective. The mutant virus makes fewer foci and the foci are weaker in appearance. The mutant middle T still associates with PI 3-kinase and shc, suggesting that the overall structure of the protein has not been disrupted. When inoculated into newborn C3H mice, the mutant virus induces fewer overall tumors with a longer latency than wild-type virus. These results indicate that this proline-rich domain in middle T antigen is important for oncogenesis in a wide variety of tissues and cell types.