Initiation of DNA replication is a tightly regulated process aimed to insure that the entire genome is replicated at the appropriate time during each cell cycle. In the human beta-globin locus, replication initiates from a region between the two genes that encode the adult subunit of hemoglobin (the beta-globin initiation region, or IR). Mammalian beta-globin loci replicate early during the S-phase of the cell cycle in pre-erythroid cells, in which the beta-globin locus is present in a euchromatin form. However, in cells that do not express globin and in which the locus is heterochromatic, these same loci replicate during the later stages of S-phase. Both early and late replication patterns utilize similar replication initiation regions. These features make the beta-globin locus an attractive model for studying the determinants of replication sites and replication timing, as well as the correlation between gene expression and DNA replication. Two genomic domains are essential for initiation of DNA replication within the locus: the initiation region (IR), and a 40 kb region upstream of the globin gene cluster known as the locus control region (LCR). The IR meets the genetic requirements for a chromosomal replicator, since it can initiate DNA replication at ectopic sites. The LCR regulates transcriptional activity and chromatin structure, and may act as a determinant of replication timing. This review will summarize recent findings characterizing the sequence requirements for initiation of DNA replication in mammalian beta-globin loci and will discuss the specific influence of the location and the chromosomal environment in regulating DNA replication at the beta-globin IR.