A chromosomal replication initiation zone was previously mapped in cell cultures to the 5' flanking DNA of the human c-myc gene. We have used an in vitro system to examine the replication of a plasmid, pNeo.Myc-2.4, containing 2.4 kb of the c-myc initiation zone. In vitro, pNeo.Myc-2.4 generated high levels of DpnI-resistant DNA above background incorporation into control plasmids. pNeo.Myc-2.4 replicated semiconservatively to produce supercoiled and relaxed plasmid monomers, and replicative intermediates. [32P]dCMP incorporated into pNeo.Myc-2.4 appeared in Okazaki fragments and low molecular weight strands which matured to full length plasmid DNA, whereas [32P]dCMP incorporated into control plasmids appeared as continuous smears on denaturing gels. Other assays also distinguished the processive replication of pNeo.Myc-2.4 from the dispersive labeling of control plasmids. A pNeo.Myc-2.4 replication time course showed a clear preference for initiation within a restriction fragment containing the c-myc DNA. Two-dimensional electrophoresis revealed that a restriction fragment bearing the c-myc origin zone generated an arc characteristic of replicative intermediates containing a central replication bubble, while vector fragments in the plasmid generated arcs of forked intermediates. Replication bubbles visualized by electron microscopy were centered within the replication initiation zone, approximately 1.4 kb upstream of c-myc promoter P1. Okazaki fragments radiolabeled during in vitro replication showed a switch in the asymmetry of template preference within the initiation zone identified by electron microscopy, two-dimensional electrophoresis and early labeling. These data show that bidirectional, semiconservative replication can originate preferentially in vitro in the 5' flanking DNA of the c-myc gene, and that replicative intermediates present at low levels can be distinguished from molecules generated by competing, repair-type processes.