DNA double strand breaks (DSBs) pose a severe hazard to the genome as erroneous rejoining of DSBs can lead to mutation and cancer. Here, we have investigated the correlation between X irradiation-induced gamma-H2AX foci, theoretically induced DSBs, and the minimal number of mis-rejoined DNA breaks (MNB) in irradiated lymphocytes obtained from two healthy humans by painting of the whole chromosome complement by spectral karyotyping. There were less gamma-H2AX foci/dose than theoretically expected, while misrepair, as expressed by MNB/gamma-H2AX focus, was similar at 0.5 and 1 Gy but 3.6-fold up at 3 Gy. Hence, our results suggest that X-Fay-induced gamma-H2AX foci in G0 lymphocyte nuclei contain more than one DSB and that the increasing number of DSBs per gamma-H2AX repair factory lead to an increased rate of misrepair. (C) 2008 Elsevier Inc. All rights reserved.