Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) can reliably sample enlarged mediastinal lymph nodes in patients with non-small cell lung cancer (NSCLC), and in practice is mostly used to sample nodes visible on CT or positron emission tomography (PET). Few data are available on the use of endoscopic procedures to stage the mediastinum in clinical stage 1 lung cancer. The aim of the present study was to determine the results of EBUS-TBNA in sampling mediastinal lymph nodes in patients with lung cancer and a radiographically normal mediastinum and no PET activity. From January 2004 to May 2007, patients highly suspicious for NSCLC with CT scans showing no enlarged lymph nodes (no node > 1 cm) and a negative PET finding of the mediastinum underwent EBUS-TBNA. Identifiable lymph nodes at locations 2r, 2L, 4r, 4L, 7, 10r, 10L, 11r, and 11L were aspirated. All patients underwent subsequent surgical staging. Diagnoses based on aspiration results were compared with those based on surgical results. One hundred patients (mean age, 52.4 years; 59 men) were included. After surgery, 97 patients (mean age, 52.9 years; 57 men) had NSCLC confirmed and were included in the analysis. In this group, 156 lymph nodes ranging 5 to 10 mm in size were detected and sampled. Malignancy was detected in nine patients but missed in one patient. Mean diameter of the punctured lymph nodes was 7.9 mm. The sensitivity of EBUS-TBNA for detecting malignancy was 89%, specificity was 100%, and the negative predictive value was 98.9%. No complications occurred. In conclusion, EBUS-TBNA can be used to accurately sample and stage patients with clinical stage 1 lung cancer and no evidence of mediastinal involvement on CT and PET. Potentially operable patients with no signs of mediastinal involvement may benefit from presurgical staging with EBUS-TBNA.