Fibroblasts in tumor tissue are thought to interact with tumor cells directly and/or indirectly and to have important roles in tumor invasion and metastasis. To characterize the phenotype of proliferating fibroblasts in pulmonary adenocarcinoma, we established short-term fibroblast cell lines from both normal bronchus and adenocarcinoma tissues obtained from the same patients and compared the gene expression profiles. Four sets of fibroblast cell lines (eight cell lines in total) were used in the analysis. Total RNA was extracted from each cell line and hybridized with 550 cancer-related RNAs blotted on a cDNA filter array. Five up-regulated genes and 12 down-regulated genes (total of 17 genes) were detected in the fibroblast cell lines from the tumor tissues compared with those from normal bronchus. Using real-time quantitative RT-PCR methods, the expression profile of each gene was examined; five genes, one up-regulated (MLH1) and four down-regulated (Cox1, FGFR4, p120, and Smad3), were confirmed. Furthermore, the protein expression levels of the five genes in the cancerous and normal tissues were examined immunohistochemically, and the up-regulation of MLH1 and the down-regulation of Cox1 in cancerous tissue were confirmed in vivo. These results indicate that the proliferating fibroblasts in pulmonary adenocarcinomas are phenotypically different from fibroblasts in normal bronchus tissues.