This study evaluated the diagnostic usefulness of tumor marker concentrations in cytologic fluids (CF) for subtyping non-small cell lung cancer (NSCLC) and assessed the relationship between fluorine-18-fluorodeoxyglucose ((18)F-FDG) uptake with serum and CF tumor marker levels. This prospective study included 88 patients diagnosed with adenocarcinoma or squamous cell carcinoma (SCC). Cytokeratin-19 fragment (CYFRA 21-1), carcinoembryonic antigen (CEA), and squamous cell carcinoma antigen (SCCA) concentrations in the CF samples were correlated with serum tumor marker concentrations, (18)F-FDG uptake, and NSCLC subtype. Fifty-eight patients were diagnosed with adenocarcinoma. Multivariate analysis revealed higher CF and serum SCCA levels; smoking status predicted SCC from adenocarcinoma. CF SCCA showed the highest accuracy (83%) in distinguishing between SCC and adenocarcinoma. CF samples obtained during routine needle aspiration biopsy procedure contain tumor marker levels sufficient to distinguish between SCC and adenocarcinoma; CF SCCA had the highest diagnostic accuracy.