By using a random mutagenesis procedure combined with a recombinant retrovirus vector, mutants of polyomavirus middle T antigen (MTAg) were generated. Three new MTAg mutants with various degrees of transformation competence were more thoroughly characterized. All of the mutants produced a stable MTAg, as assessed by metabolic labeling or immunoblotting, and each mutant possessed wild-type levels of associated tyrosine kinase activity and associated phosphatidylinositol-3 (PI-3) kinase activity. One of these mutants, with a substitution of leucine for proline at amino acid 248 of MTAg (248m) was completely transformation defective, as measured in a focus-forming assay. Furthermore, the pattern of phosphorylation of 248m in vivo was identical to that of wild-type MTAg, and the kinetics of association of MTAg with an 85-kilodalton protein, the putative PI kinase, was not altered. Similarly, the pattern of PI derivatives obtained in an in vitro kinase assay was not altered by the substitution at amino acid 248. Since the single base pair mutation at amino acid 248 resulted in an MTAg that was completely transformation defective despite possessing wild-type levels of kinase activities, this suggests that neither tyrosine kinase nor PI-3 kinase activity nor the combination of both are sufficient for transformation by MTAg.