To study correlations between cellular transformation and the biochemical properties of polyomavirus middle T antigen, middle T cDNAs have been derived from the polyomavirus mutants dl1015, dl23, and NG59b and have been introduced into rodent fibroblast cell lines by using a retrovirus vector. It was found that all three mutants are completely defective in inducing growth in soft agar but possess a range of activities in assays of focus formation on cell monolayers. Furthermore, when assays of middle T antigen-associated kinase activities were performed in vitro, a correlation between the level of associated phosphatidylinositol kinase activity and the ability of mutant middle T antigens to induce focus formation was observed. However, the association of this activity with middle T antigen does not appear to be sufficient to bring about full transformation, since the middle T antigen derived from dl1015 is completely defective for soft-agar growth but is associated with a level of phosphatidylinositol kinase activity which is comparable to that of the wild type. Therefore, some other unidentified middle T antigen function may also be required for full transformation.