The adenovirus E1b-58kd tumor antigen has been detected in a physical association with a 54 kilodalton cellular protein in adenovirus-transformed mouse cells. Antibody specific for the E1b-58kd protein coimmunoprecipitates a 54 kd protein from transformed, but not from productively infected, cells. Monoclonal antibody specific for the cellular 54 kd protein coimmunoprecipitates the adenovirus E1b-58kd protein from transformed cell extracts. The same or closely related cellular 54 kd protein, associated with the adenovirus E1b-58kd protein, was present in the SV40 large T antigen-54 kd complex previously detected in SV40-transformed mouse cells. The identity of the 54 kd protein is based on the immunological specificities of the anti-54 kd monoclonal antibodies and partial peptide maps of the 54 kd protein associated with the adenovirus and SV40 tumor antigens. The adenovirus E1b-58kd-54 kd complex, like the SV40 large T antigen-54 kd complex, is heterogeneous in size or mass. While all of the cellular 54 kd protein in the adenovirus-transformed cell extract is found in a complex with the E1b-58kd protein, some of the viral 58 kd antigen is detected in a form not associated with the 54 kd protein. The fact that the adenovirus and Sv40 tumor antigens, both required for transformation, can be found in physical association with the same cellular protein in a transformed cell is a good indication that these two diverse viral proteins share some common mechanisms or functions.