Lung cancer metastasis is a complicated process in which multiple stages and multiple genes are involved. There is an urgent need to use new molecular biology techniques to get more systematic information and have a general idea of the molecular events that take place in lung cancer metastasis. The object of this study was to construct the subtracted cDNA libraries of different metastatic potential lung cancer cell lines, NL9980 and L9981, which were established and screened from human lung large cell carcinoma cell line, WCQH-9801.   The forward and reverse subtracted cDNA libraries were constructed in the large cell lung cancer cell lines NL9980 and L9981 with the same heredity background but different metastatic potential, by suppression subtractive hybridization (SSH). The positive clones were preliminarily screened by blue-white colony and precisely identified by PCR. The forward and reverse subtracted libraries were screened and identified by dot blot so as to obtain the clones corresponding to gene segments with differential expression. DNA sequencing was performed to analyze the sequences of differential expression segments, which were then searched and compared using the Basic Local Alignment Search Tool from The National Center for Biotechnology Information NCBI BLAST tools. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed to confirm the differential expressed genes both on RNA and protein levels.   The forward and reverse subtracted cDNA libraries of the different large cell lung cancer cell lines with metastatic potential were successfully constructed. With blue-white colony and dot blot, 307 positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained. Fifty-five clones were successfully sequenced in the forward subtracted library while 31 clones were successfully sequenced in the reverse subtracted library. One new expressed sequence tag (EST) segment was identified from the reverse subtracted cDNA library and was successfully submitted to GenBank and embodied by GenBank. For the differentially expressed genes between L9981 and NL9980 screened by SSH, four genes, ANXA2, KRT18, ACTG1 was upregulated in L9981 cells compared to NL9980 cells. Annexin A2 (which was encoded by ANXA2), γ-actin (which was encoded by ACTG1), and aldose reductase (which was encoded by AKR1B1) proteins were upregulated in L9981 cells compared to NL9980 cells by western blotting.   The forward and reverse subtracted cDNA libraries of different metastatic potential large cell lung cancer cell lines were successfully constructed by SSH. A series of genes have been screened out to have significantly different expression levels between lung cancer cell lines NL9980 and L9981. A new EST segment that may represent a new metastasis-related gene has been identified. Consistent with the result of SSH, both quantitative real-time RT-PCR and western Blotting confirmed the upregulation of ANXA2, ACTG1 and AKR1B1 in lung cancer cell line L9981 compared with NL9980. These three genes may play important roles in lung cancer metastasis. © 2011 Tianjin Lung Cancer Institute and Blackwell Publishing Asia Pty. Ltd.