Many new carcinogenesis-related genes of esophageal cancer, including neutrophil gelatinase-associated lipocalin (NGAL), had been cloned when the differentially expressed genes in the process of the esophageal epithelial cell transforming to carcinoma were being looked for. In these genes there might be some control mechanisms of functional networks. In order to further study the network relationships of the genes, a cDNA library from a esophageal cancer cell line(SHEEC),which had been established, for yeast-hybrid system will be constructed. Total RNA was extracted from SHEEC by Trizol reagent and PolyA⁺mRNA was purified from total RNA by Oligotex mRNA Kit. cDNA was synthesized using SuperScript^TM Choice System For cDNA Synthesis Kit. The quality of synthesized cDNA was detected by chemical light method using PolyA⁺ mRNA probe. The double-strand cDNA was ligated into the EcoRⅠ site of pGADT₇ vector and the cDNA library from the esophageal cancer cell line for yeast-hybrid system was constructed. The titer of the amplified cDNA library was 1.19×10⁹ cfu/ml，The inserted fragment size of recombinants was from 0.5 kb to 6.0 kb and the percentage of recombinant clones was about 50％. NF-κB element binding factors were screened from this cDNA library by yeast one-hybrid technic and more than 360 clones were obtained on the SD/-his/-leu/［＋15 mmol/L 3-AT］. Plasmids of 91 clones which were larger than 2 mm in diameter were isolated from yeast and transformed to E.coli 30 positive recombinants had been obtained since plasmids were extracted from E.coli and digested by EcoRⅠ and validated by yeast one-hybrid assay, 9 clones of them were sequenced and the sequence was compared with GenBank/BLAST database. Results showed that protein product of some genes obviously conformed to consensus sequence of NF-κB element binding domain of p65 or p50 in the key sites of amino acid residues. These results determined that the human esophageal cancer cDNA library for yeast-hybrid system had a higher quality.