The cellular response to DNA-damaging agents involves the activation of cell cycle checkpoints. Checkpoints provide a transient delay in cell cycle progression, presumably to allow time for the cell to repair the damage. A most important checkpoint, active in the G1 phase of the cell cycle, is mediated by the p53 tumor suppressor gene product. To investigate the role of downstream components of the cell cycle machinery in p53-mediated G1 arrest, the possible involvement of the RB gene product was examined. Rb and p53 proteins were studied by immunoprecipitation and Western blotting experiments in the presence and absence of DNA-damaging treatment. The phosphorylation status of Rb was altered following DNA damage in p53 wild-type cell lines, but was not altered in p53 mutant cell lines, nor in cell lines where p53 function was abrogated by viral gene products. These findings indicate that Rb probably plays a role in the activation of the p53-mediated checkpoint.