Activating mutations of EGFR are found frequently in a subgroup of patients with non-small cell lung cancer (NSCLC) and are highly correlated with the response to gefitinib and erlotinib. In the present study, we searched for mutations of EGFR, HER2 and KRAS in 264 resected primary NSCLC from Japanese patients and determined whether there is a correlation between genetic alterations of these genes and clinicopathological factors, together with 85 tumors that we reported previously. EGFR mutations were found in 102 of the total 349 tumors, and seven tumors had two missense mutations. Reverse transcriptionpolymerase chain reaction of EGFR and subsequent subcloning analyses identified that the double mutations occurred in the same allele. Although the prognosis for patients with advanced NSCLC has not improved in the last 20 years, (3) the molecular and biological characteristics of lung cancer have been studied intensively (4,5) and many molecules associated with the pathogenesis of lung cancer have been identified as promising new targets for lung cancer therapy.(6) Among these molecules, EGFR, one of the ERBB family of receptors, is thought to be a promising target because of its frequent overexpression (range 40 -80%) in NSCLC (7) and its relationship with poor prognosis.(8) Small molecules of EGFR-TKI, such as gefitinib and erlotinib, have been developed, examined for the treatment of NSCLC, and shown to have antitumor activity.(9) Several clinical studies have revealed that Japanese female never-smoking patients with adenocarcinoma have a higher response rate to gefitinib. (10,11) Recently, activating mutations of the TK domain of EGFR were found in a unique subgroup of NSCLC and were highly correlated with the response to EGFR-TKI.(12-14) Subsequent analyses using a large series of NSCLC confirmed somatic mutations in the similar subgroup of patients as known clinical predictors of EGFR-TKI sensitivity.(15-18) Moreover, recent studies have shown that increased copy numbers of the EGFR gene are associated with a better response to EGFR-TKI. (17,19) HER2, another member of the ERBB family, can heterodimerize with EGFR and has also been shown to have somatic mutations in the TK domain in several cancers including NSCLC. (20,21) However, the involvement of HER2 alterations in the pathogenesis of NSCLC is not clearly understood.After earlier reporting 17 mutations of EGFR in 85 NSCLC, (22) we further extended our analysis with another 264 NSCLC. In a total of 349 tumors, we found 102 with EGFR mutation, six with HER2 mutation, and 21 with KRAS mutation. We also identified seven tumors with double mutations of EGFR and eight tumors with both mutation and amplification of EGFR. We therefore investigated the double genetic event of EGFR in detail. Our results provide new insights into the involvement of alterations of the receptor TK family in NSCLC development.