A stable cell line expressing the bovine papillomavirus E1 protein (C2E1) was compared with an E1 minus control line (CNEO) to study the effects of E1 protein on host cell growth. C2E1 and CNEO cells were synchronized either at mitosis or at the G1/S boundary by the cell cycle inhibitors nocodazole and mimosine, respectively. After release from the drug-induced cell cycle block, the progression through the succeeding stages of the cell cycle was temporally monitored using flow cytometry. In addition, incorporation of bromodeoxyuridine (BrdUrd) was used to determine precisely the time of initiation of DNA synthesis in C2E1 and CNEO cells after release from drug-induced cell cycle arrest. Expression of E1 protein decreased the duration of G1 phase and increased S and G2 phase durations without affecting the overall cell doubling time. In conjunction with the increase in G2 phase duration, histone H1 kinase activity was prolonged during the G2 to M phase transition in C2E1 cells, which suggested that E1 protein may affect the mechanisms which ensure proper timing of kinase inactivation. During the G1 to S phase transition in C2E1 cells, the timing of appearance and abundance of cyclin D1 were altered compared to CNEO cells, while cyclin E levels were unaffected. Consequently, E1 protein may affect G1 phase duration through a cyclin D1-dependent pathway. Finally, a subpopulation of cells with a greater than G2 DNA content (>G2 DNA), and which was still capable of incorporating BrdUrd, was shown to exist only in the E1-expressing cell line. These combined results demonstrate that the viral replication protein E1 has the potential to influence the host cell environment significantly, which may contribute to pathogenesis and viral persistence.