At supramolecular resolution, DNA synthesis begins at preferred replication origins in the chromosomes of metazoan cells. To characterize one of these origins in detail, the initiation of replication was examined in the HeLa c-myc origin. Polymerase chain reaction (PCR) amplification of size-fractionated nascent chromosomal DNAs revealed multiple replication initiation sites over a 12-kb region spanning the c-myc origin, including the transcribed region and the 5' and 3' flanking DNA of the gene. Two of the start sites for chromosomal replication occurred inside a 2.4-kb region of the origin that exhibits autonomously replicating sequence (ARS) activity. When a plasmid containing the 2.4-kb ARS region was transfected into HeLa cells, PCR mapping of nascent plasmid DNA confirmed that the plasmid replicated semiconservatively and autonomously and that replication did not initiate at random sites but rather began at multiple sites in a limited zone overlapping the c-myc DNA insert. Within the resolution of the PCR assay, the same sites that were used in the chromosomal c-myc origin were used in the 2.4-kb ARS fragment. The locations of replication start sites determined by PCR are considered in the context of other functional and structural elements of the c-myc origin.