The N-terminal part of the mouse polyomavirus T antigens contains a highly conserved segment (-LLELLKL-), including amino acid residues 13 to 19. The sequence motif is predicted to form alpha helix I in the DnaJ domain of the T antigens. Four mutants with conservative substitutions of amino acid residues 13 and 14 were constructed. Of the four substitutions, L13M, L13I, L13V, and L14V, only L13V resulted in a phenotypic change. In transfected mouse cells, L13V large T antigen showed a more than 100-fold-reduced viral DNA synthesis. The viral replication could not be rescued by cotransfection of the cells with DNA expressing small t antigen or a large T antigen truncated at the C terminus that would compensate for a defect in host cell stimulation. In contrast to the effect on DNA replication, the L13V substitution in large T antigen did not prevent complex formation with Hsc70 and the Rb protein. Also, the activity of the protein in transactivation of transcription from the adenovirus E2 promoter was unimpaired, showing that the transcription factor E2F was released from pRb. The L13V substitution also caused a defect in small t antigen. However, this phenotypic change was due to protein instability. In contrast, middle T antigen with the L13V substitution remained stable and functional in cellular transformation. Together, the data show that the effect of the L13V substitution did not abrogate the Hsc70 interaction of the DnaJ domain. However, it is possible that the substitution of amino acid residue 13 affected specific DnaJ functions of large T antigen.