The treatment of patients with non-small cell lung cancer (NSCLC) depends on the stage. Positron emission and computed tomography (CT) scans can identify suspicious lymph nodes that require biopsy. We prospectively evaluated the yield and accuracy of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in sampling mediastinal lymph nodes and compared its accuracy to that of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and CT in staging NSCLC. A consecutive series of patients with suspicious nodes on PET or CT scan in the posterior mediastinal lymph node stations (#5, 7, 8, or 9) were prospectively evaluated by EUS-FNA. The reference standard included thoracotomy with complete lymphadenectomy in patients with lung cancer or if EUS-FNA was benign, repeat clinical imaging, or long-term follow-up. There were 104 patients (63 men) with 125 lesions (117 lymph nodes, 8 left adrenal glands) who underwent EUS-FNA. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of EUS-FNA were 92.5%, 100%, 100%, 94%, and 97%, respectively. EUS-FNA was more accurate and had a higher positive predictive value than the PET or CT (p < 0.001) scan in confirming cancer in the posterior mediastinal lymph nodes. EUS-FNA documented metastatic cancer to the left adrenal in all 4 patients with advanced disease. No deaths resulted from EUS-FNA. One patient experienced self-limited stridor. EUS-FNA is a safe, accurate, and minimally invasive technique that improves the staging of patients with NSCLC. It is more accurate and has a higher predictive value than either the PET scan or CT scan for posterior mediastinal lymph nodes. © 2005 by The Society of Thoracic Surgeons.