After infection of L cells with the DNA-defective temperature-sensitive (ts) mutant 6389 of vaccinia virus, [3H]thymidine incorporation into cytoplasmic DNA is inhibited at 39 degrees, but resumes upon shiftdown to 32 degrees, the permissive temperature. Following a 30-min lag period DNA synthesis is linear and contingent upon continuous protein synthesis. Sedimentation analysis of nascent DNA labeled during 10 to 60-min pulses revealed that the mutant molecules are produced at a slower rate, but are approximately the same size as those of wild-type vaccinia, synthesized under the same circumstances. During more prolonged incubation beyond 60 min, labeled DNA molecules sedimenting more rapidly than mature, full-length virus genomes are observed. The integration of mutant DNA into mature virions is less rapid than that of the wide-type DNA. Upon extraction from the virosomes, the ts6389 DNA sediments as both genome-size and larger, faster sedimenting DNA. Upon treatment with restriction endonucleases, the ts6389 virosomal DNA exhibited an additional fragment after separation on agarose gels, perhaps as a consequence of fusion between the terminal fragments of the molecule. Taken together these observations suggest that concatemeric intermediates are formed during vaccinia DNA replication. By measuring the radioactivity incorporated into the fragments and subfragments of the molecules labeled during the first round of replication, the initiation site of replication can be localized to a region within the terminal 150 bp.