Physical interactions between human cytomegalovirus (HCMV) immediate-early (IE) proteins and key cell cycle regulatory proteins have been suggested as a mechanism whereby this herpesvirus modifies cellular control of proliferation. Observed similarities to interactions of other DNA virus proteins (human papillomavirus type 16 E6 and E7, simian virus 40 large T antigen, and adenovirus type 5 E1A and E1B) with cell cycle modulatory proteins such as p53 and Rb have suggested that HCMV IE proteins may likewise alter the G1-to-S phase transition. The IE2 region gene product IE86 has been shown to specifically bind p53, potentially modifying p53 G1 checkpoint function. To examine this possibility, p53-mediated G1 arrest in the presence of IE86 was assessed. Retroviral constructs were created to facilitate the stable expression of IE86 and IE72, another IE protein implicated in HCMV-mediated alteration of cell cycle progression. Western analysis and immunoprecipitation confirmed IE protein expression and binding of IE86 to p53, respectively. Chloramphenicol acetyltransferase assays examining the ability of IE86 to repress activity from the HCMV major IE promoter or activate the HCMV early promoter for the 2.2-kb class of RNAs demonstrated the functional integrity of the IE86 protein. Induction of DNA damage in normal, uninfected fibroblasts (FB) or FB expressing IE86 by actinomycin D (Act D) resulted in increased p53 levels, a predominance of the hypophosphorylated form of Rb, and increased expression of both p21(CIP1/WAF1) and mdm-2. Fluorescence-activated cell sorting revealed that both uninfected and IE86-expressing FB experienced dramatic G1 arrest following exposure to Act D. The clear demonstration of these p53-dependent responses in the presence of IE86 indicates that binding to this viral protein does not compromise the ability of p53 to elicit growth arrest following DNA damage.