We evaluated the usefulness of a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) method for detecting occult tumor cells in histologically malignant-negative lymph nodes resected from patients with non-small cell lung cancer. First, we examined the relationship between tumor cell number and carcinoembryonic antigen (CEA) mRNA copy number using a PCR method with a cancer cell line (A549) in a serial dilution study. Next, we evaluated the relationship between nodal metastatic area size and CEA mRNA copy number using lymph nodes with histologically proven metastasis in a serial slice study. On the basis of those results, we performed RT-PCR analyses with 28 primary tumors and 211 lymph nodes from 28 patients who underwent a lobectomy with systematic node dissection. Our results in the serial dilution study showed that the detectable limitation by quantitative RT-PCR was 25-100 neoplastic cells and 20-100 CEA mRNA copy numbers. In the serial slice study, we found a correlation between CEA mRNA copy number and nodal metastatic area. In the clinical samples, amplification of CEA mRNA was obtained with all 28 primary tumors and 13 of the lymph nodes with metastasis shown by hematoxylin-eosin staining. Furthermore, 52 (25%) of 211 histologically negative lymph nodes and the specimens from 14 (64%) of the 22 pN0 patients revealed a significant level of CEA mRNA. These results indicate that micrometastases, which are not detectable with conventional examinations, can be detected by the present method of RT-PCR for CEA mRNA in a proportion of patients with resected pN0 non-small cell lung cancer.