Mediastinal lymph nodes are the most common site of tumor spread in non-small cell lung cancer (NSCLC). We hypothesized that micrometastatic disease could be detected by reverse transcription-polymerase chain reaction (RT-PCR) for expression of human telomerase reverse transcriptase (hTERT) in mediastinal lymph nodes and that a minimally invasive technique (endoscopic ultrasound-guided fine-needle aspiration [EUS-FNA]) is capable of sampling lymph nodes for PCR analysis without surgery. Mediastinal lymph nodes were sampled with EUS-FNA in patients with NSCLC and negative control subjects undergoing EUS for benign disease. Total RNA was harvested from samples, and RT-PCR was performed to detect telomerase gene expression. RNA was available from 87 of 100 lymph node aspirates from 39 patients with NSCLC and from 12 negative control patients. hTERT was expressed in 0 of 14 negative control lymph nodes in 18 of 57 pathologically negative lymph nodes from cancer patients and in 10 of 16 pathologically positive lymph nodes (p < 0.05). Five of 18 (28%) patients with no pathologically evident mediastinal disease expressed telomerase in at least one lymph node. Minimally invasive EUS-FNA with RT-PCR is capable of detecting expression of cancer specific mRNA in lymph nodes. Approximately one-third of pathologically negative mediastinal lymph nodes in NSCLC patients express hTERT mRNA. The clinical significance of this observation is yet to be determined.