Eukaryotic initiation of DNA replication is a tightly regulated process. In the yeasts, S-phase-specific cyclin Cdk1 complex as well as Dfb4-Cdc7 kinase phosphorylate the initiation factors Sld2 and Sld3. These factors form a ternary complex with another initiation factor Dbp11 in their phosphorylated state, and associate with the origin of replication. This complex mediates the loading of Cdc45. A second complex called GINS and consisting of Sld5 and Psf1, 2 and 3 is also loaded onto the origin during the initiation process, in an interdependent manner with the Sld2/Sld3/Dpb11 complex. Both complexes cooperate in the recruitment of the replicative DNA polymerases, thus executing the initiation and subsequent establishment of the replication fork. Cdc45 and GINS are essential, well-conserved factors that are retained at the elongating replication fork. They form a stable helicase complex with MCM2-7 and mediate its contact to the replicative DNA polymerases. In contrast, the Sld2/Sld3/Dpb11 complex critical for the initiation is not retained by the elongating replication fork. Sld2 displays limited homology to the amino-terminal region of RecQL4 helicase, which may represent its metazoan orthologue, whereas Sld3 homologues have been identified only in fungi. Dbp11 and its fission yeast homologue Cut5 are members of a large family of BRCT-containing proteins including human TopBP1 and fruit fly Mus101. Similar principles of regulation apply also to human initiation of DNA replication, despite obvious differences in the detailed mechanisms. The regulatory initiation cascade is intimately intertwined with the cell cycle apparatus as well as the checkpoint control.