Mutations in the epidermal growth factor receptor (EGFR) gene have been identified in patients with non-small-cell lung cancer (NSCLC) who respond to anilinoquinazoline EGFR inhibitors. Despite initial responses to these inhibitors, most patients eventually develop resistance. This study reports a case of a patient with EGFR-mutant, gefitinib-responsive NSCLC who developed resistance after two years of complete remission. DNA sequencing of the tumor biopsy at relapse revealed a second point mutation, resulting in a threonine-to-methionine amino acid change at position 790 of EGFR. Structural modeling and biochemical studies showed that this second mutation led to gefitinib resistance. The patient was a 71-year-old former smoker with advanced, moderately differentiated adenocarcinoma of the lung. He had a deletion, delL747-S752, in the EGFR gene, which is associated with responsiveness to gefitinib. Despite treatment with carboplatin, taxanes, and gemcitabine, the patient had a clinical and radiographic response to gefitinib monotherapy. After 24 months of complete remission, his symptoms worsened, and computed tomography revealed progressive lung abnormalities consistent with relapse. At this point, gefitinib was stopped, and a transbronchial aspirate and biopsy were obtained. Both cytologic and pathological analysis confirmed the presence of recurrent, moderately differentiated adenocarcinoma. The patient's relapse was hypothesized to be due to an acquired, second mutation in the EGFR gene that conferred resistance to gefitinib. The EGFR tyrosine kinase domain was resequenced in the second biopsy specimen, revealing a second mutation, T790M. The T790M mutation was shown to confer resistance to gefitinib by altering the binding site of the drug, leading to steric hindrance. Structural modeling confirmed that the T790M mutation causes a structural change similar to that seen in the BCR-ABL tyrosine kinase domain, leading to resistance to imatinib. The T790M mutation also allows the activation of EGFR, leading to clinical resistance. The study highlights the importance of repeated biopsies in clinical studies of novel targeted therapies and suggests that alternative EGFR inhibitors or inhibitors of downstream targets such as phosphatidylinositol 3'-kinase or STAT5 may be necessary for patients with EGFR-mutant tumors with acquired resistance to anilinoquinazoline inhibitors.Mutations in the epidermal growth factor receptor (EGFR) gene have been identified in patients with non-small-cell lung cancer (NSCLC) who respond to anilinoquinazoline EGFR inhibitors. Despite initial responses to these inhibitors, most patients eventually develop resistance. This study reports a case of a patient with EGFR-mutant, gefitinib-responsive NSCLC who developed resistance after two years of complete remission. DNA sequencing of the tumor biopsy at relapse revealed a second point mutation, resulting in a threonine-to-methionine amino acid change at position 790 of EGFR. Structural modeling and biochemical studies showed that this second mutation led to gefitinib resistance. The patient was a 71-year-old former smoker with advanced, moderately differentiated adenocarcinoma of the lung. He had a deletion, delL747-S752, in the EGFR gene, which is associated with responsiveness to gefitinib. Despite treatment with carboplatin, taxanes, and gemcitabine, the patient had a clinical and radiographic response to gefitinib monotherapy. After 24 months of complete remission, his symptoms worsened, and computed tomography revealed progressive lung abnormalities consistent with relapse. At this point, gefitinib was stopped, and a transbronchial aspirate and biopsy were obtained. Both cytologic and pathological analysis confirmed the presence of recurrent, moderately differentiated adenocarcinoma. The patient's relapse was hypothesized to be due to an acquired, second mutation in the EGFR gene that conferred resistance to gefitinib. The EGFR tyrosine kinase domain was resequenced in the second biopsy specimen, revealing a second mutation, T790M. The T790M mutation was shown to confer resistance to gefitinib by altering the binding site of the drug, leading to steric hindrance. Structural modeling confirmed that the T790M mutation causes a structural change similar to that seen in the BCR-ABL tyrosine kinase domain, leading to resistance to imatinib. The T790M mutation also allows the activation of EGFR, leading to clinical resistance. The study highlights the importance of repeated biopsies in clinical studies of novel targeted therapies and suggests that alternative EGFR inhibitors or inhibitors of downstream targets such as phosphatidylinositol 3'-kinase or STAT5 may be necessary for patients with EGFR-mutant tumors with acquired resistance to anilinoquinazoline inhibitors.