Purpose: Epidermal growth factor receptor (EGFR) and other members of the ErbB family of receptor tyrosine kinases (RTK) mediate autocrine growth regulation in a wide spectrum of human tumor cells. We have previously demonstrated that in stably transfected mammary carcinoma cells a dominant negative (DN) mutant of EGFR, EGFR-CD533 is a potent inhibitor of EGFR and its cytoprotective signaling after exposure to ionizing radiation. In the present study, we further investigate the capacity of a genetic approach, using replication-incompetent adenovirus (Ad)-mediated transfer of EGFR-CD533 (Ad-EGFR-CD533), to enhance the radiosensitivity in vitro of four cell lines representative of three major cancer phenotypes., Methods and Materials: The cell lines MDA-MB-231 and T-47D mammary carcinoma, A-431 squamous carcinoma, and U-373 MG malignant glioma cells were used. The ErbB expression profiles and the EGFR tyrosine phosphorylation (Tyr-P) levels following irradiation were quantified by Western blotting. The relative radiosensitivities of tumor cells were assessed by standard colony formation assays after infection with control vector (Ad-LacZ) or Ad-EGFR-CD533., Results: The expression profiles demonstrated varying levels of EGFR, ErbB2, ErbB3, and ErbB4 expression. The overexpression of EGFR-CD533 after infection with Ad-EGFR-CD533 completely inhibited the radiation-induced stimulation of EGFR Tyr-P relative to the immediate 2.4- to 3.1-fold increases in EGFR Tyr-P in control infected cells (Ad-LacZ). Ad-EGFR-CD533-infected cells demonstrated significant (p < 0.001) radiosensitization over a range of radiation doses (1-8 Gy), yielding dose-enhancement ratios (DER) between 1.4 and 1.7. This radiosensitization was maintained under conditions of repeated radiation exposures, using 3 x 2 Gy, yielding DERs of 1.6 and 1.7 for MDA-MB-231 and U-373 cells, respectively., Conclusions: Overexpression of EGFR-CD533 significantly sensitizes human carcinoma and glioma cells to single and repeated radiation exposures irrespective of their ErbB expression levels. Therefore, transduction of human tumor cells with EGFR-CD533 holds promise as a gene therapeutic approach for the radiosensitization of neoplastic cells that are growth-regulated by EGFR or other ErbB receptors.