PURPOSE: The relationship between plasma concentration and antitumor activity of gefitinib was assessed in patients with advanced non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. PATIENTS AND METHODS: Plasma trough levels of gefitinib were measured on days 2 (D2) and 8 (D8) by high-performance liquid chromatography in 31 patients. Plasma concentrations of gefitinib were also measured 10 h after the first administration in 21 of these patients to calculate the elimination half-life of gefitinib. RESULTS: The median trough levels were: 197 ng/ml 10 h from the first administration of gefitinib; 113 ng/ml on D2; and 358 ng/ml on D8. The median D8/D2 ratio was 2.709, and the median elimination half-life was 15.7 h. The median progression-free survival (PFS) was 273 days, and the median overall survival (OS) was 933 days. A high D8/D2 ratio was significantly correlated with better PFS, though the plasma trough levels on D2 and D8 were not significantly related to PFS. The elimination half-life was not a significant factor for PFS, but it was significantly correlated with high-grade adverse events. Pharmacokinetic parameters were not significantly correlated with OS. CONCLUSIONS: A high D8/D2 ratio, but not elimination half-life, might be a predictor of better PFS in patients with NSCLC harboring EGFR mutations treated with gefitinib. On the other hand, long elimination half-life was related to high-grade adverse events in these patients. Clinical Trial Registration UMIN000001066.