We have constructed a polyomavirus mutant genome which exhibits an increased immortalization potential when transfected into primary rat embryo fibroblasts. The mutation is a 30-base-pair deletion (nucleotides 1367 through 1396) that inactivates the transforming potential of middle T but activates some of the properties of large T associated with neoplastic transformation. Unlike the wild-type large T, the mutant large T can fully complement polyoma middle T in the tumorigenic process in vivo as well as in the transformation of primary cells in vitro. The activity of the mutant can be explained by its inability to replicate in cells and, hence, its inability to exert a cytopathic effect after gene transfer at high multiplicity. A recombinant which encodes the middle and small T antigens, but not the large T antigen, can also elicit a fully transformed phenotype when introduced into primary rat fibroblasts. These results confirm previous observations from this laboratory indicating that two, and not three, viral gene functions are required for polyomavirus-mediated oncogenic transformation.