Cell cycle arrest in response to DNA damage is important for the maintenance of genomic integrity in higher eukaryotes. We have previously reported the novel p53-dependent S-phase checkpoint operating in mouse zygotes fertilized with irradiated sperm. In the present study, we analysed the detail of the p53 function required for this S-phase checkpoint in mouse zygotes. The results indicate that ATM kinase is likely to be indispensable for the p53-dependent S-phase checkpoint since the suppression was abrogated by inhibitors such as caffeine and wortmannin. However, ATM phosphorylation site mutant proteins were still capable of suppressing DNA synthesis when microinjected into sperm-irradiated zygotes lacking the functional p53, suggesting that the target of the phosphorylation is not p53. In addition, the suppression was not affected by alpha-amanitin, and p53 protein mutated at the transcriptional activation domain was also functional in the suppression of DNA synthesis. However, p53 proteins mutated at the DNA-binding domain were devoid of the suppressing activity. Taken together, the transcription-independent function of p53 associated with the DNA-binding domain is involved in the S-phase checkpoint in collaboration with yet another unidentified target protein(s).