Background: Gefitinib, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has activity against approximately 10% of unselected non–small-cell lung cancer (NSCLC) patients. Phosphatidylinositol 3′-kinase (PI3K)/Akt and Ras/Raf/mitogen-activated protein kinase (MAPK), the two main EGFR-signaling pathways, mediate EGFR effects on proliferation and survival. Because activation of these pathways is dependent on the phosphorylation status of the components, we evaluated the association between phosphorylation status of Akt (P-Akt) and MAPK (P-MAPK) and gefitinib activity in patients with advanced NSCLC. Methods: Consecutive patients (n = 106) with NSCLC who had progressed or relapsed on standard therapy received gefitinib (250 mg/day) until disease progression, unacceptable toxicity, or patient refusal. P-Akt and P-MAPK positivity was determined with immunohistochemistry using tumor tissues obtained before any anticancer treatment. Association of P-Akt and time to progression was determined by univariable and multivariable analyses. All statistical tests were two-sided. Results: Of the 103 evaluable patients, 51 (49.5%) had tumors that were positive for P-Akt, and 23 (22.3%) had tumors that were positive for P-MAPK. P-Akt–positivity status was statistically significantly associated with being female (P<.001), with never-smoking history (P = .004), and with bronchioloalveolar carcinoma histology (P = .034). Compared with patients whose tumors were negative for P-Akt, patients whose tumors were positive for P-Akt had a better response rate (26.1% versus 3.9%; P = .003), disease control rate (60.9% versus 23.5%; P<.001), and time to progression (5.5 versus 2.8 months; P = .004). Response rate, disease control rate, and time to progression did not differ according to P-MAPK status. The multivariable analysis showed that P-Akt positivity was associated with a reduced risk of disease progression (hazard ratio = 0.58, 95% confidence interval = 0.35 to 0.94). Conclusions: Patients with P-Akt–positive tumors who received gefitinib had a better response rate, disease control rate, and time to progression than patients with P-Akt–negative tumors, suggesting that gefitinib may be most effective in patients with basal Akt activation.