Background: Many patients with non–small-cell lung cancer (NSCLC) who achieve radiographic responses to treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib have somatic mutations in the EGFR tyrosine kinase domain. However, little is known about the efficacy of cetuximab, an antibody against the EGFR extracellular domain, in EGFR mutant NSCLC. Methods: NSCLC cell lines carrying wild-type EGFR (A549, H441, and H1666) or mutant EGFR (H3255, DFCILU-011, PC-9, and HCC827) were treated with various dilutions of gefitinib or cetuximab relative to maximal achievable serum concentration. Cell growth was analyzed by the MTS assay, with differences between dose–response curves analyzed nonparametrically. Apoptosis was analyzed by propidium iodide staining and immunoblotting for PARP. Phosphorylation of EGFR and the downstream signaling components ERK1/2 and Akt were analyzed by immunoblotting. Statistical tests were two-sided. Results: Growth of NSCLC lines with wild-type EGFR was slightly (A549 and H441) or moderately (H1666) inhibited by gefitinib and cetuximab, and the effects of the two agents were similar. Both agents also induced no (H441) or moderate (H1666) apoptosis in NSCLC cells with wild-type EGFR. By contrast, gefitinib was statistically significantly more effective than cetuximab at inhibiting growth of EGFR mutant cells (H3255: P = .003, DFCILU-011: P = .011, and PC-9: P = .003), and gefitinib-treated EGFR mutant cells had higher levels of apoptosis than cetuximab-treated cells (mean fold increase in apoptosis by 1 μ M of gefitinib and 10 μg/mL of cetuximab relative to control, H3255: 8.3 [95% confidence interval {CI} = 4.8 to 11.8] and 2.1 [95% CI = 2.0 to 2.2], respectively, P = .025; DFCILU-011: 5.7 [95% CI = 5.1 to 6.3] and. 0.9 [95% CI = 0.3 to 1.5], respectively, P <.001). Gefitinib treatment decreased EGFR, ERK1/2, and Akt phosphorylation in EGFR mutant cell lines whereas cetuximab had relatively little effect. Both gefitinib and cetuximab inhibited the growth of HCC827 cells, but gefitinib inhibited growth to a greater extent ( P = .003). Conclusions: EGFR mutations in NSCLC cells are associated with sensitivity to gefitinib but not to cetuximab.