P155 is a polyomavirus mlt mutant with normal transforming ability but impaired tumorigenic potential. The mutation, a 12-bp deletion (nucleotides 1348-1359), removes amino acids 372 to 375 from middle T and affects its ability to function in tumorigenesis (C. Gelinas, S. Masse, and M. Bastin, 1984, J. Virol. 51, 242-246). We used deletion loop mutagenesis to introduce point mutations within the wild-type sequence spanned by the P155 deletion. A mutant phenotype resembling that of P155 could be produced by as little as one alanine to valine substitution at residue 373. The mutants were impaired in their ability to induce tumors in rats but they could still transform established cell lines or primary fibroblasts in culture. To define the biochemical defect, we examined the mutant middle T antigen both for association with pp60c-src, the cellular src gene product, as well as its pattern of phosphorylation. No obvious differences explaining the phenotype were observed. The mutant middle T associated with, and activated pp60c-src, but exhibited a slightly altered pattern of phosphorylation, presumably because of additional sites on the middle T protein.