SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.