The observation that transcriptionally active genes generally replicate early in S phase and observations of the interaction between transcription factors and replication proteins support the thesis that promoter elements may have a role in DNA replication. To test the relationship between transcription and replication we constructed HeLa cell lines in which inducible green fluorescent protein (GFP)-encoding genes replaced the proximal approximately 820-bp promoter region of the c-myc gene. Without the presence of an inducer, basal expression occurred from the GFP gene in either orientation and origin activity was restored to the mutant c-myc replicator. In contrast, replication initiation was repressed upon induction of transcription. When basal or induced transcription complexes were slowed by the presence of alpha-amanitin, origin activity depended on the orientation of the transcription unit. To test mechanistically whether basal transcription or transcription factor binding was sufficient for replication rescue by the uninduced GFP genes, a GAL4p binding cassette was used to replace all regulatory sequences within approximately 1,400 bp 5' to the c-myc gene. In these cells, expression of a CREB-GAL4 fusion protein restored replication origin activity. These results suggest that transcription factor binding can enhance replication origin activity and that high levels of expression or the persistence of transcription complexes can repress it.