E2F transcription factors play pivotal roles in controlling the expression of genes involved in cell viability as well as genes involved in cell death. E2F1 is an important constituent of this protein family, which thus far contains eight members. The interaction of E2F1 with its major regulator, retinoblastoma protein (Rb), has been studied extensively in the past two decades, concentrating on the role of E2F1 in transcriptional regulation and the role of Rb in cell replication and cancer formation. Additionally, the effect of viral infections on E2F1/Rb interactions has been analyzed for different viruses, concentrating on cell division, which is essential for viral replication. In the present study, we monitored E2F1-Rb interactions during human herpesvirus 6A (HHV-6A) and HHV-6B infections of SupT1 T cells. The results have shown the following dramatic alterations in E2F1-Rb pathways compared to the pathways of parallel mock-infected control cultures. (i) The E2F1 levels were elevated during viral infections. (ii) The cellular localization of E2F1 was dramatically altered, and it was found to accumulate both in the cytoplasmic and nuclear fractions, as opposed to the strict nuclear localization seen in the mock-infected cells. (iii) Although E2F1 expression was elevated, two exemplary target genes, cyclin E and MCM5, were not upregulated. (iv) The Rb protein was dephosphorylated early postinfection, a trait that also occurred with UV-inactivated virus. (v) Infection was associated with significant reduction of E2F1/Rb complexing. (vi) HHV-6 infections were accompanied by cell cycle arrest. The altered E2F1-Rb interactions and functions might contribute to the observed cell cycle arrest.