We have isolated a novel human lung‐specific gene, LUNX (lung‐specific X protein), by differential‐display mRNA analysis. The full‐length cDNA contained 1,015 nucleotides including an open reading frame of 768 nucleotides encoding 256 amino acids. We localized the gene to chromosomal region 20p11.1‐q12 by radiation hybrid mapping. Using an RT‐PCR assay specific for LUNX mRNA, 35 non‐small‐cell lung‐cancer (NSCLC) tumors and 0 of 16 normal lymph nodes were positive. Furthermore, LUNX mRNA expression was enhanced in 26 (84%) of 31 NSCLC tumors vs. corresponding cancer‐free lung tissues by semi‐quantitative analyses with multiplex RT‐PCR. We assessed the possibility of LUNX mRNA as a molecular marker for detection of micrometastasis in dissected lymph nodes obtained from 20 patients with NSCLC tumors. LUNX mRNA was detected in 16 (80%) of 20 histologically positive lymph nodes and 21 (25%) of 84 histologically negative lymph nodes. Comparative analyses of the conventional histological examination and the RT‐PCR detection assay for LUNX mRNA showed that the detection rate of metastases in lymph nodes by the RT‐PCR assay was higher in 12 and consistent in 6 of the total 20 NSCLC patients. We demonstrate that the LUNX RT‐PCR assay is a potential diagnostic method for detection of micrometastases in lymph nodes of NSCLC patients. © 2001 Wiley‐Liss, Inc.