To search a novel factor possibly involved in the regulation of DNA replication for eukaryotic cells, we fractionated the Xenopus egg extract frequently used as a cell free DNA replication system, and obtained a single polypeptide of 50 KDa on SDS-polyacrylamide gel electrophoresis. Data of the amino acid sequence reduced from cloned gene of this protein revealed that this might be a Xenopus homolog of human PA2G4 that was known as a proliferation-associated protein or a cell cycle-regulated protein. Recombinant mouse PA2G4 protein also showed the inhibitory effect on the DNA replication reaction in Xenopus egg extract with the single-stranded DNA template. These results strongly suggest that PA2G4 may have an important role on the regulation of DNA replication in eukaryotic cells.