Epidermal growth factor receptor (EGFR) and HER-2 are associated with a poor prognosis in various cancers, including prostate cancer. Inhibition of these receptors may provide a treatment for hormone-refractory prostate cancer. The presence of HER-2 (Western blot) and EGFR (5830 fmol/mg protein, ligand-binding assay) was assessed in the hormone-refractory human prostate cancer cell line, DU-145. Cells were exposed to the selective EGFR-TKI (EGFR tyrosine kinase inhibitor) gefitinib ('Iressa; ZD1839) and/or the HER-2-targeted monoclonal antibody trastuzumab ('Herceptin'), for 96 h. Irradiation (RX) at 6 Gy the dose causing 50% growth inhibition, was applied 48 h after the start of drug treatment. There was a dose-related effect on cell survival for both ZD1839 and trastuzumab treatments. Combining ZD1839 and trastuzumab led to less than additive effects on cell survival. Chou and Talalay representations further characterised this less than additive effect on cell survival. The application of ZD1839 led to a marked elevation in the level of the negative regulator of cell division, p27. The ZD1839-trastuzumab combination had less of an impact on p27 expression compared with the effect of ZD1839 treatment alone. The lowest expression of the apoptotic-related protein, Bax, was observed in the presence of the drug combination. There was a significant interaction (synergism) between RX and either ZD1839 or trastuzumab treatments. In contrast, the drug combination with RX resulted in antagonistic cytotoxic effects. These results indicate an antagonistic interaction between EGFR and HER-2 targeting and provide molecular mechanisms supporting this observation. Data from DU-145 cells suggest that dual targeting of EGFR and HER-2 may be inappropriate for the treatment of hormone-refractory prostate cancer, especially in the context of their combination with RX.