DNA replication is a dynamic process that requires the precise coordination of numerous cellular proteins. At the core of replication in eukaryotic cells are three DNA polymerases, Pol α, Pol δ, and Pol e, which function cooperatively to ensure efficient and high-fidelity genome replication. These enzymes are members of the B family of DNA polymerases, characterized by conserved amino acid motifs within the polymerase active sites. Pol α is a DNA polymerase of moderate fidelity that lacks 3′→5′ exonuclease activity, while Pols δ and e are processive, high-fidelity polymerases with functional 3′→5′ exonuclease activities. Each polymerase exists as a holoenzyme complex of a large polymerase catalytic subunit and several smaller subunits. The Pol α holoenzyme possesses primase activity, which is required for de novo synthesis of RNA–DNA primers at replication origins and at each new Okazaki fragment. In one model of eukaryotic DNA replication, Pol e functions in leading strand DNA synthesis, while Pol δ functions primarily in lagging strand synthesis. This chapter discusses the biochemical properties of eukaryotic replicative polymerases and how biochemical properties shape their functional roles in replication initiation, replication fork elongation, and the checkpoint responses.