Transcriptional regulation of the bacteriophage T4 late genes requires the participation of three DNA polymerase accessory proteins that are encoded by T4 genes 44, 62, and 45, and that act at an enhancer-like site. Transcriptional activation by these DNA replication proteins also requires the function of an RNA polymerase-bound coactivator protein that is encoded by T4 gene 33 and a promoter recognition protein that is encoded by T4 gene 55. Transcriptional activation in DNA constructs, in which the enhancer and a T4 late promoter can be segregated on two rings of a DNA catenane, has now been analyzed. The ability of an interposed DNA-binding protein to affect communication between the enhancer and the promoter was also examined. Together, these experiments demonstrate that this transcription-activating signal is conveyed between its enhancer and a T4 late promoter by a DNA-tracking mechanism. Alternative activation mechanisms relying entirely on through-space interactions of enhancer-bound and promoter-bound proteins are excluded.