Microscopically visible gammaH2AX foci signify the presence of DNA double-strand breaks (dsbs) in irradiated cells. However, large foci are also observed in untreated tumour cells, and high numbers reduce the sensitivity for detecting drug or radiation-induced DNA breaks. SW756 cervical carcinoma cells that express about 50 gammaH2AX foci per cell (i.e., equivalent to the number of breaks produced by about 2Gy) showed similar numbers of dsbs as C33A cells that exhibit fewer than three foci per cell. The possibility that differences in numbers of these endogenous foci could be explained by genomic instability perhaps related to misrepair was examined. For 17cell lines selected from the panel of NCI-60 tumor cells previously characterized for karyotypic complexity [A.V. Roschke, G. Tonon, K.S. Gehlhaus, N. McTyre, K.J. Bussey, S. Lababidi, D.A. Scudiero, J.N. Weinstein, I.R. Kirsch, Karyotypic complexity of the NCI-60 drug-screening panel, Cancer Res. 63 (2003) 8634-8647], there was a significant trend (r=0.6) for cell lines with greater numbers of structural or numerical chromosomal rearrangements to show a higher background expression of gammaH2AX. Moreover, cells from this panel with wild-type p53 showed a significantly lower background level of gammaH2AX than cells with mutant p53. To confirm the importance of p53 expression, endogenous and radiation-induced gammaH2AX expression were analyzed using four isogenic SKOV3 cell lines varying in p53 function. Again, higher gammaH2AX expression was found in SKOV3 cell lines expressing mutant p53 compared to wild-type p53. HFL-1 primary lung fibroblasts showed a progressive increase in gammaH2AX as they moved towards senescence, confirming the importance of telomere instability in the development of at least some gammaH2AX foci. Therefore, the explanation for high endogenous levels of gammaH2AX in some tumor cells appears to be multifactorial and may be best described as a consequence of chromatin instability.