The structure of replication origins in metazoans is only nominally similar to that in model organisms, such as Saccharomyces cerevisiae. By contrast to the compact origins of budding yeast, in metazoans multiple elements act as replication start sites or control replication efficiency. We first reported that replication forks diverge from an origin 5′ to the human c-myc gene and that a 2.4-kb core fragment of the origin displays autonomous replicating sequence activity in plasmids and replicator activity at an ectopic chromosomal site. Here we have used clonal HeLa cell lines containing mutated c-myc origin constructs integrated at the same chromosomal location to identify elements important for DNA replication. Replication activity was measured before or after integration of the wild-type or mutated origins using PCR-based nascent DNA abundance assays. We find that deletions of several segments of the c-myc origin, including the DNA unwinding element and transcription factor binding sites, substantially reduced replicator activity, whereas deletion of the c-myc promoter P1 had only a modest effect. Substitution mutagenesis indicated that the sequence of the DNA unwinding element, rather than the spacing of flanking sequences, is critical. These results identify multiple functional elements essential for c-myc replicator activity.