A heat-stable DNA-binding protein with subunits of about 53 kilodaltons (kDal) was purified from two virally transformed human cell lines (Epstein-Barr virus-positive Raji and Namalwa) and two mouse tumor cell lines (methylcholanthrene-induced Meth A sarcoma and TA3 mammary carcinoma). All four 53kDal proteins showed closely related total amino acid compositions, similar peptide maps, and identical NH2-terminal amino acid sequences for 20 residues. These 53-kDal proteins are therefore evolutionarily highly conserved, independent of whether they originate from virally or chemically transformed cells. The NH2-terminal sequence and the protein chain as a whole are not hydrophobic; however, some unexpected residue distributions were observed. Comparisons with other proteins reveal no clear sequence similarity with known tumor antigen structures, homologous immunoglobulins, or some other proteins of known sequence. Epstein-Barr virus-determined nuclear antigen also appears to have a different NH2-terminal sequence. Thus, the results show that the 53-kDal proteins represent a unique protein type with little species variation; this finding suggests that these proteins must perform an important common function in different transformation systems.