Although various mutations of the epidermal growth factor receptor (EGFR) gene, most commonly L858R (exon 21) and short exon 19 deletions, have been identified to confer sensitivity toward EGFR tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, it is not known if there are mutations that may result in differential activities of the two inhibitors. We describe a 70-year-old Japanese American woman diagnosed with stage IV non-small-cell lung cancer (NSCLC) with rib metastasis. While receiving treatment with the EGFR small molecule TKI erlotinib, she progressed and developed new brain metastases. She failed further chemotherapy treatments and subsequently developed symptomatic extensive leptomeningeal carcinomatosis associated with diplopia, hemiparesis, weight loss, and incontinence. Monotherapy gefitinib 250 mg daily was initiated, and she showed striking response both clinically and radiographically within the first few weeks. Using laser microdissection (LMD), we performed genomic DNA extraction and EGFR gene sequencing from the enriched tumor cells in her pretreatment tumor biopsy specimen and tumor cells found in her cerebrospinal fluid. Two heterozygous somatic EGFR mutations, L858R (exon 21) and E884K (exon 22), were identified in both specimens. In vitro transfection and biochemical studies revealed that the novel E884K mutation confers opposite effects in sensitivity to the two EGFR inhibitors. EGFRE884K and EGFRL858R+E884K enhanced the sensitivity of the mutated receptor to gefitinib inhibition. Conversely, the E884K mutation resulted in decreased responsiveness of the receptor to erlotinib, and it significantly abrogated the drug sensitivity conferred by L858R (EGFRL858R+E884K). This study demonstrates that it is possible to have differential response to alternative EGFR TKIs. This also represents the first report of a response of leptomeningeal metastases to EGFR inhibition by small molecule inhibitor gefitinib alone in NSCLC. Further structural studies of the mutant EGFR are warranted to improve individualized targeted therapy and small molecule inhibitors' design in lung cancer in the future.