Initiation and completion of DNA replication defines the beginning and ending of S phase of the cell cycle. Successful progression through S phase requires that replication be properly regulated and monitored to ensure that the entire genome is duplicated exactly once, without errors, in a timely fashion. Given the immense size and complexity of eukaryotic genomes, this presents a significant challenge for the cell. As a result, DNA replication has evolved into a tightly regulated process involving the coordinated action of numerous factors that function in all phases of the cell cycle. We will review our current understanding of these processes from the formation of prereplicative complexes in preparation for S phase to the series of events that culminate in the loading of DNA polymerases during S phase. We will incorporate structural data from archaeal and bacterial replication proteins and discuss their implications for understanding the mechanism of action of their corresponding eukaryotic homologues. We will also describe the concept of replication licensing which protects against genomic instability by limiting initiation events to once per cell cycle. Lastly, we will review our knowledge of checkpoint pathways that maintain the integrity of stalled forks and relay defects in replication to the rest of the cell cycle. © 2005 Nature Publishing Group. All rights reserved.