Monoclonal antibodies, one against the adenovirus type 5 E1B 55-kDa protein and one against the E1B 21-kDa protein, have been isolated and shown to recognize the same proteins as antitumor sera. Immunofluorescence studies with these monoclonal antibodies on transformed cells containing the complete adenovirus early region 1, showed that the E1B 21-kDa protein is localized in the perinuclear region. The E1B 55-kDa protein is localized in a number of different sites: a strong fluorescence is observed in a discrete body in the cytoplasm close to the nucleus, a moderate fluorescence is found in cell-cell contacts, and a weak staining in the cytoplasm. The cellular p53 antigen, which is associated with the E1B 55-kDa protein, is also found in the discrete cytoplasmic body, but not, or only in small amounts at the cell-cell contacts. However, p53 is not seen in the cytoplasm outside the discrete body, but the nucleus is weakly positive. The nature of the discrete cytoplasmic body was investigated further by electron microscopy and was found to be composed of a cluster of 8-nm filaments. The diameter of the filaments is similar to that of cytoskeletal intermediate filaments. However, staining with antibodies against the various intermediate filament proteins did not show a significant reaction with the cluster, while vimentin intermediate filaments could be demonstrated in the cells in a typical cytoskeletal pattern. It was also shown that the cluster is not composed of incorrectly aggregated tubulin.