We used the deletion loop mutagenesis procedure to direct point mutations into a small region of the polyomavirus genome, at 0.97 map units, affecting the structure of both the middle and large T antigens. We describe the construction of six middle T mutants which have retained the ability to transform rat cells in culture and four large T mutants, three of which are deficient in viral DNA replication and capable of immortalizing primary rat embryo fibroblasts very efficiently.