Using a technique developed recently to detect DNA polymerase activity in situ after NaDodSO4 gel electrophoresis (Spanos, A., Sedgwick, S. G., Yarranton, g. T., Hübscher, U. & Banks, G. R. (1981) Nucleic Acids Res. 9, 1825-1839), we present evidence that a high Mr (greater than or equal to 125,000) polypeptide is responsible for chromosomal DNA replication in prokaryotes, lower eukaryotes and high eukaryotes. Not only extracts from Escherichia coli, Ustilago maydis, Drosophila melanogaster, rat neurones, calf thymus, human fibroblast, and HeLa cells possess such high Mr activities, but also highly purified E. coli DNA polymerase III core enzyme, U. maydis DNA polymerase, and D. melanogaster embryo and calf thymus DNA alpha polymerases. The evidence that these activities are responsible for chromosomal DNA replication is genetical (E. coli, U. maydis, and D. melanogaster); also, the high Mr activity disappears from rat neurones during differentiation from an actively dividing precursor cell to a postmitotically mature neurone. Furthermore, when limited proteolysis is allowed to occur, a defined and remarkably similar pattern of intermediate Mr activities is generated in lower eukaryotic and high eukaryotic extracts and, to some extent, in prokaryotic extracts. In higher eukaryotic extracts, a low Mr activity of approximately 35,000 is also generated. Protease inhibitors can retard formation of these catalytically active proteolytic fragments. We propose that the replicative DNA polymerase complex of both prokaryotes and eukaryotes contains a high Mr polypeptide responsible for chain elongation which might be conserved during evolution and which is extremely sensitive to proteolytic cleavage.