DNA polymerases δ and ɛ (Polδ and Polɛ) are widely thought to be the major DNA polymerases that function in elongation during DNA replication in eukaryotic cells. However, the precise roles of these polymerases are still unclear. Here we comparatively analysed DNA replication inXenopusegg extracts in which Polδ or Polɛ was immunodepleted. Depletion of either polymerase resulted in a significant decrease in DNA synthesis and accumulation of short nascent DNA products, indicating an elongation defect. Moreover, Polδ depletion caused a more severe defect in elongation, as shown by sustained accumulation of both short nascent DNA products and single‐stranded DNA gaps, and also by elevated chromatin binding of replication proteins that function more frequently during lagging strand synthesis. Therefore, our data strongly suggest the possibilities that Polδ is essential for lagging strand synthesis and that this function of Polδ cannot be substituted for by Polɛ.