Epidermal growth factor receptor (EGFR) overexpression has been implicated in the development of head and neck squamous cell carcinomas (HNSCC) and represents a potential therapeutic target for this disease. We have reported previously that growth inhibitory concentrations of IFN-alpha enhance the expression and activity of EGFR and that this effect could represent an escape mechanism to the growth inhibition and apoptotic cell death induced by IFN-alpha. In this study, we investigate whether the combination of IFN-alpha and gefitinib (Iressa, AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom), a selective EGFR tyrosine kinase inhibitor, might have a cooperative antitumor effect on HNSCC-derived cell lines. The interaction of IFN-alpha and gefitinib was evaluated in vitro on HNSCC-derived cell lines by median drug effect analysis calculating a combination index with CalcuSyn software and in vivo by using HNSCC xenografts in nude mice. The mechanism of gefitinib and IFN-alpha interactions was also studied by analysis of cell cycle kinetics, apoptosis assays, and Western blotting of EGFR signal transduction components. Simultaneous exposure to gefitinib and IFN-alpha produced synergistic antiproliferative and proapoptotic effects compared with single drug treatment. Furthermore, daily treatment of gefitinib (50 mg/kg p.o.) in combination with an IFN-alpha regimen (50,000 units s.c. three times weekly) induced tumor growth delay and increased survival rate on established HNSCC xenografts in nude mice. Moreover, the concomitant treatment with gefitinib suppressed the stimulation of extracellular signal-regulated kinase phosphorylation/activity induced by IFN-alpha both in vitro and in vivo. The observed cooperative antitumor effects could be, at least in part, explained by the inhibition exerted by gefitinib of an IFN-alpha-induced EGF-dependent survival pathway, which involves extracellular signal-regulated kinase activation. These results provide a rationale for the clinical evaluation of gefitinib in combination with IFN-alpha in HNSCC.