We have determined that HSV causes rapid and large increases in cell-cycle-regulated free E2F and S-phase p107/E2F DNA binding activities in asynchronous cultures of C33A cells. Induction occurred by 4 hr postinfection and coincided with the appearance of viral encoded immediate-early and delayed-early proteins, i.e., when viral DNA replication normally commences. No increase in E2F activities occurred when cells were infected with viruses expressing mutant regulatory proteins ICP4 or ICP27, or mutant replication proteins ICP8, pol or helicase, or when cells were infected with wild-type virus in the presence of inhibitors of DNA synthesis. In contrast, ICP8 mutant-infected cells contained elevated amounts of NF kappa B activity equivalent to WT virus, no induction of Sp1 relative to WT virus, and reduced ATF/CREB activity relative to WT virus. Results of transient expression assays with E2F-responsive reporters indicated that the net effect of induction of both active (free E2F) and repressive (p107/E2F) complexes was a decrease in AdE2 promoter activity and an increase in c-myc promoter activity. Taken together these results suggest that HSV can cause unscheduled changes in the amount and functional status of a cell-cycle-regulated transcription factor. These results are discussed in light of possible roles for viral-induced alterations in E2F, especially as related to imposing or overriding cell-cycle checkpoints.