Despite improvements in the detection and use of biomarkers, including epidermal growth factor receptor, ERCC1, and p16, the 5-year survival rate with non-small cell lung cancer remains at 15%. This suggests that additional biomarkers are needed to better prognosticate clinical course and guide therapeutic approaches. Previous studies showed that increased levels of aspartyl (asparaginyl)-beta-hydroxylase and a highly related molecule, humbug, correlate with clinical course and survival with hepatic, biliary, pancreatic, and colon carcinomas. We now characterize the prognostic use of aspartyl (asparaginyl)-beta-hydroxylase/humbug immunoreactivity in different subtypes of non-small cell lung cancer. Tissue microarrays including 375 paraffin-embedded non-small cell lung cancers (195 adenocarcinomas; 18 bronchioloalveolar carcinomas; 113 squamous cell carcinomas; and 49 large cell carcinomas) were immunostained with FB50 monoclonal antibody, which recognizes human aspartyl (asparaginyl)-beta-hydroxylase/humbug. Immunoreactivity (intensity and distribution) in neoplastic cells were scored under code, and data were subjected to univariate and Cox multivariate analyses, adjusting for age, stage, and treatment. High levels of FB50 immunoreactivity were more often detected in adenocarcinomas (28% for adenocarcinoma, 17% for bronchioloalveolar carcinoma), compared with squamous cell carcinomas (10%) and large cell carcinomas (10%). Univariate analysis demonstrated inverse relationships between intensity of FB50 immunoreactivity and survival with squamous cell carcinoma (P = .004), and a strong trend with respect to large cell carcinoma (P = .057). Cox multivariate test showed that FB50 immunoreactivity (P = .025), clinical stage (P = .029), and tumor size (P = .0001) were all independent predictors of survival with squamous cell carcinoma. High levels of FB50 immunohistochemical staining correlate with poor prognosis in non-small cell lung cancer, particularly squamous cell carcinoma subtype. Therefore, FB50 immunoreactivity may be useful in defining patient subsets that are likely to benefit from adjuvant therapy.