Checkpoint kinase 2 (CHK2) is a major effector of the DNA damage response pathway and although its mechanism of activation has been well studied, the attenuation of its activity following DNA damage has not been explored. Here, we identify the B'alpha subunit of protein phosphatase 2A (PP 2A) as a CHK2 binding partner and show that their interaction is modulated by DNA damage. B'alpha binds to the SQ/TQ repeat region of CHK2, which is a target of ATM phosphorylation. The induction of DNA double-strand breaks by gamma irradiation as well as treatment with doxorubicin causes dissociation of the B'alpha and CHK2 proteins. This dissociation correlates with an increase in the ATM-dependent phosphorylation of CHK2 at serines 33 and 35 in the SQ/TQ region. Indeed, mutating these sites to mimic phosphorylation increases the dissociation after irradiation. PP 2A negatively regulates CHK2 phosphorylation at multiple sites, as well as its kinase activity. These data reveal a novel mechanism for PP 2A to keep CHK2 inactive under normal conditions while also allowing for a rapid release from this regulation immediately following DNA damage. This is followed by a subsequent reconstitution of the PP 2A/CHK2 complex in later time points after damage, which may help to attenuate the signal.