The animal cell genome is organized into a series of replicons with an average size of 50-300 kilobases; each of these units is characterized by its own origin of replication which serves as the point of initiation for DNA synthesis. In animal viruses, origin usage can be regulated by cis-acting elements, and in some cases, replication may be cell-type specific. Little is known, however, about the organization and control of endogenous tissue-specific gene replication. To understand this process, we have used a replication direction assay to examine DNA fragments covering more than 200 kilobases of the human beta-like globin domain, and have identified a single bidirectional origin located upstream of the beta-globin itself. This locus is used to initiate DNA synthesis in expressing cells, where the globin domain replicates early, and in non-expressing cells, which are characterized by late replication of the same region. Deletion of this origin sequence, as occurs in the haemoglobin Lepore syndrome, cancels bidirectional DNA synthesis at this site and leads to a striking reversal of replication direction upstream to the locus. This represents the first genetic proof of the existence of specific, discrete origins of replication in animal cells.