A common cellular response to DNA damage is cell cycle arrest. This checkpoint control has been the subject of intensive genetic investigation, but the biochemical mechanism that prevents mitosis following DNA damage is unknown. In Schizosaccharomyces pombe, as well as vertebrates, the timing of mitosis under normal circumstances is determined by the balance of kinases and phosphatases that regulate inhibitory phosphorylation of Cdc2. In S. pombe, the phosphorylation occurs on tyrosine-15. This method of mitotic control is also used in S. pombe to couple mitosis with completion of DNA replication, but the role of Cdc2 tyrosine phosphorylation in the Chk1 kinase-mediated DNA damage checkpoint has remained uncertain. We show that, in contrast to recent speculation, the G2 DNA damage checkpoint arrest in S. pombe depends on the inhibitory tyrosine phosphorylation of Cdc2 carried out by the Wee1 and Mik1 kinases. Furthermore, the rate of Cdc2 tyrosine dephosphorylation is reduced by irradiation. This result implicates regulation of Cdc2 tyrosine dephosphorylation, mainly carried out by the Cdc25 tyrosine phosphatase, as an important part of the mechanism by which the DNA damage checkpoint induces Cdc2 inhibition and G2 arrest.