The development of novel immunotherapy strategies for non-small cell lung cancer (NSCLC) will be facilitated by the identification of tumor-specific targets. Although the epidermal growth factor receptor (EGFR) is overexpressed in many cases of NSCLC, its wide distribution in normal tissue may limit its suitability as an immunotherapeutic target. However, mutations within the EGFR that are unique to malignancies may provide specific targets for immunotherapeutic intervention. For example, one mutant form, the type III deletion mutant of the EGFR, that has been identified in glioblastomas contains a novel peptide sequence in its extracellular domain which is detectable by anti-peptide antisera. In this study, the prevalence of this type of mutation of the EGFR in NSCLC was determined. Thirty-two frozen sections of primary NSCLC were examined by immunocytochemistry to determine the presence of native and mutated EGFR. Native EGFR was overexpressed in 12 of the 32 sections and a diffuse cellular distribution of the EGFR type III deletion mutation was identified in five (16%) of the specimens (2 of 13 squamous, 2 of 2 mixed, 0 of 10 adenocarcinoma, and 1 of 7 undifferentiated). This mutated EGFR was not detected in sections of normal breast, lung, skin, ovary, colon, kidney, endometrium, and placenta. The type III EGFR deletion mutant, expressed in some cases of NSCLC, may be a molecularly defined, tumor-specific antigen in lung cancer.