A 77-year-old male with a history of smoking (B.I. 800) was diagnosed with lung adenocarcinoma. DNA analysis revealed a mutation of the epidermal growth factor receptor (EGFR) gene in exon 19 deletion in his bronchoscopic transbronchial biopsy specimens by the Cycleave method. During observation only, as wished by the patient, the tumor grew gradually and pleural effusion occurred. He was diagnosed with malignant pleural effusion, but EGFR gene mutation was not observed in that specimens by the PNA-LNA PCR clamp method. Treatment with gefitinib was started and the tumor size and pleural effusion remarkably decreased. After 4 weeks of treatment with gefitinib, a re-biopsy was done. We could find adenocarcinoma by bronchoscopy, but cancer cells were not observed in pleural effusion. When analysis of the EGFR gene was conducted, mutation was not observed in the pulmonary tissue by the RT–PCR method. Nowadays, we can use several EGFR gene mutation detection methods using the PCR technique, and each is almost equal in quality. But some reports showed that different results were received from different methods. We probably thought the result of pretreatment specimen from pleural effusion was false negative. The EGFR tyrosine kinase inhibitor (EGFR-TKI) such as gefitinib is a key drug for mutated-EGFR non-small-cell lung cancer. Therefore, we should avoid the loss of treatment opportunity with EGFR-TKI because of false-negative results.