Phosphorylated H2AX is considered to be a biomarker for DNA double-strand breaks (DSB), but recent evidence suggests that γH2AX does not always indicate the presence of DSB. Here we demonstrate the bimodal dynamic of H2AX phosphorylation induced by ionizing radiation, with the second peak appearing when G2/M arrest is induced. An increased level of γH2AX occurred in mitotic cells, and this increase was attenuated by DNA-PKcs inactivation or Chk2 depletion, but not by ATM inhibition. The phosphorylation-mimic CHK2-T68D abrogated the attenuation of mitotic γH2AX induced by DNA-PKcs inactivation. Thus, the DNA-PKcs/CHK2 pathway mediates the mitotic phosphorylation of H2AX in the absence of DNA damage. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.