To clarify the relationship between various functions of the polyomavirus large T antigen and the contribution of this oncogene toward neoplastic transformation, we have analyzed the properties of mutants with in-frame deletions in the second large T exon. dl45, dl96, and dl97 have retained the ability to immortalize primary rat embryo fibroblasts and to trans-activate viral promoters. dl8, dl23, and dl300, which are deficient immortalization, are also deficient in transactivation. However, a newly constructed mutant, designated dl141, which is deficient in immortalization, is still able to trans-activate both the polyoma and SV40 late promoters. This indicates that the ability to trans-activate promoters is not sufficient to confer on the large T antigen the ability to immortalize primary cells.