The epidermal growth factor receptor (EGFR), which is over-expressed in tumors cells of epithelial origin is one of the determinants of tumor responses to ionizing radiation. Recently, it has been shown that higher EGFR expression levels lead to higher tumor resistance to radiation therapy through the activation of cell proliferation and survival pathways. In this study, a raster-scan imaging technique known as Number and Brightness (N&B) analysis has been employed to demonstrate the nuclear translocation of EGFR in living cells under a variety of experimental conditions. About 80% of wild type (WT) EGFR translocated to the nucleus after γ-irradiation while the L858R and ΔE746-E750 mutant EGFR did not. Subsequently, the effects of γ-irradiation together with an EGFR-blocking antibody (cetuximab) were monitored simultaneously in the same cell lines expressing EGFR and its mutants. In the combined radiation and cetuximab treatment, about 26 % of WT were translocated to the nucleus, while the L858R and ΔE746-E750 mutant EGFR did not. These results are consistent with findings attained by standard molecular techniques and support the hypothesis that a cytosolic pool of EGFR exists that cannot be accessed by cetuximab and can therefore contribute to treatment resistance.