Mouse cells transformed by simian virus 40 often contain virus-coded tumor antigens distinct from those synthesized in productively infected permissive cells. The SV3T3 C120 cell line produces no large T-antigen of apparent molecular weight 94,000 but instead a super T-antigen of apparent molecular weight 145,000. We used recombinant DNA techniques to isolate the template for this super T-antigen and determined its structure by DNA sequencing. The integrated viral early transcription unit contains an in-phase, perfect tandem duplication of 1,212 base pairs. Transfer hybridization and endonuclease S1 mapping experiments were performed to elucidate the structures of the stable, cytoplasmic mRNAs of SV3T3 C120 cells, mRNAs of 3.9 and 3.6 kilobases, containing the small t- and large T-antigen splices, respectively, were transcribed from the internally duplicated early transcription unit. We showed by in vitro translation that these mRNAs encode small t-antigen and the super T-antigen of molecular weight 145,000. Peptide mapping studies of the SV3T3 C120 super T-antigen were consistent with its being derived from an internally duplicated template, since the protein has methionine and cysteine tryptic fingerprints virtually identical to those of normal large T-antigen, with certain methionine peptides present in greater than one molar yield.