Purpose: Epidermal growth factor receptor (EGFR) genotyping is now standard in the management of advanced lung adenocarcinoma, as this biomarker predicts marked benefit from treatment with EGFR tyrosine kinase inhibitors (TKI). EGFR exon 19 insertions are a poorly described family of EGFR mutations, and their association with EGFR-TKI sensitivity in lung adenocarcinoma is uncertain. Experimental Design: Patients with lung cancers harboring EGFR exon 19 insertions were studied. The predicted effects of the insertions on the structure of the EGFR protein were examined, and EGFR exon 19 insertions were introduced into Ba/F3 cells to assess oncogenicity and in vitro sensitivity to EGFR-TKIs. In patients receiving TKI, response magnitude was assessed with serial computed tomographic (CT) measurement. Results: Twelve tumors harboring EGFR exon 19 insertions were identified; patients were predominately female (92%) and never-smokers (75%). The 11 specimens available for full sequencing all showed an 18- bp insertion that resulted in the substitution of a Pro for Leu at residue 747. The mutant EGFR transformed the Ba/F3 cells, which were then sensitive to EGFR-TKI. Six patients with measurable disease received TKI and five had a response on serial CT. Conclusions: EGFR exon 19 insertions are a newly appreciated family of EGFR-TKI-sensitizing mutations, and patients with tumors harboring these mutations should be treated with EGFR-TKI. While these mutations may be missed through the use of some mutation-specific assays, the addition ofPCR product size analysis to multigene assays allows sensitive detection of both exon 19 insertion and deletion mutations.