Epidermal growth factor receptor (EGFR) has been extensively targeted in the treatment of non–small cell lung cancer, producing responses in a small number of patients. To study the role of ligand expression in mediating response to EGFR antagonism, we injected NCI-H441 [EGFR and EGF/transforming growth factor-α (TGF-α) positive] or PC14-PE6 (EGFR positive and EGF/TGF-α negative) human lung adenocarcinoma cells into the lungs of nude mice. We randomized the mice to receive treatment with the EGFR tyrosine kinase inhibitors gefitinib or AEE788 or vehicle. Treatment of mice bearing NCI-H441 but not PC14-PE6 lung tumors resulted in a significant reduction in primary tumor growth, pleural effusion, and lymph node metastasis. Immunohistochemical analyses revealed that NCI-H441 and PC14-PE6 cells expressed EGFR but that the expression of EGF/TGF-α was high in NCI-H441 cells and very low in PC14-PE6 cells. Consequently, EGFR was activated in both tumor and tumor-associated endothelial cells in the NCI-H441 tumors but not in the PC14-PE6 tumors. Antagonism of EGFR signaling by treatment of mice with AEE788 decreased proliferation and increased apoptosis of both tumor cells and tumor-associated endothelial cells in NCI-H441 tumors but not in PC14-PE6 tumors. However, after transfection of PC14-PE6 cells with TGF-α, lung tumors derived from the transfected cells expressed and activated EGFR in both tumor and tumor-associated endothelial cells and tumors responded to treatment with AEE788. Collectively, these results strongly suggest that the response of human lung cancers growing orthotopically in mice to the inhibition of EGFR signaling is determined by ligand (EGF/TGF-α) expression by tumor cells. Our findings provide an additional explanation for the susceptibility of lung cancers to treatment with EGFR tyrosine kinase inhibitors. [Mol Cancer Ther 2007;6(10):2652–63]