DNA replication in the human -globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the -globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5HS2-5 in the normal chromosomal context of the human -globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5HS2-5 in the classically defined LCR do not control replication in the human -globin locus. Recent studies also show that targeted deletion of 5HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.The eukaryotic genome is divided into independently regulated domains that initiate DNA replication from defined sequences at specific times during S phase. Control mechanisms exist to specify both the sites of replication initiation (origins) and the temporal order of replication throughout the genome. Although great progress has been made in understanding the control of DNA replication in viruses, bacteria, and yeasts, we are only beginning to understand the control of DNA replication in higher eukaryotes. Over a dozen origins have been mapped in metazoans (reviewed in reference 10), and it appears that DNA replication in these organisms, in contrast to prokaryotes and lower eukaryotes, does not depend only on origin-proximal sequences, but is subject to long-distance regulation as well. The human -globin locus is one of several loci in which this long-distance regulation has so far been demonstrated; sequences over 50 kb from the origin of replication are necessary for proper replication initiation and replication timing of the locus. Because of the tools available with which to make genetic modifications and perform functional analyses, the human -globin locus offers an excellent opportunity to dissect possible replication control mechanisms.A single bidirectional origin, located in the vicinity of the -globin gene, is used to replicate the human -globin locus ( Fig. 1) (1, 35). Although this origin is used in both erythroid and nonerythroid cells, replication timing follows patterns of gene activity and chromatin structure: in erythroid cells, the globin genes are transcribed, the locus is gen...