The inhibition of replicative DNA synthesis that follows DNA damage may be critical for avoiding genetic lesions that could contribute to cellular transformation. Exposure of ML-1 myeloblastic leukemia cells to non-lethal doses of the DNA damaging agents, γ-irradiation or actinomycin D, causes a transient inhibition of replicative DNA synthesis via both G₁ and G₂ arrests. Levels of p53 protein in ML-1 cells and in proliferating normal bone marrow myeloid progenitor cells increase and decrease in temporal association with the G₁ arrest. In contrast, the S-phase arrest of ML-1 cells caused by exposure to the anti-metabolite, cytosine arabinoside, which does not directly damage DNA, is not associated with a significant change in p53 protein levels. Caffeine treatment blocks both the G₁ arrest and the induction of p53 protein after γ-irradiation, thus suggesting that blocking the induction of p53 protein may contribute to the previously observed effects of caffeine on cell cycle changes after DNA damage. Unlike ML-1 cells and normal bone marrow myeloid progenitor cells, hematopoietic cells that either lack p53 gene expression or overexpress a mutant form of the p53 gene do not exhibit a G₁ arrest after γ-irradiation; however, the G₂ arrest is unaffected by the status of the p53 gene. These results suggest a role for the wild-type p53 protein in the inhibition of DNA synthesis that follows DNA damage and thus suggest a new mechanism for how the loss of wild-type p53 might contribute to tumorigenesis.