Immortalization is an early and essential step or human carcinogenesis which is associated with alterations in gene expression and regulation. Suppression subtractive hybridization (SSH) was successfully performed to identify immortalization-associated genes upregulated in SV40-immortalized lung fibroblasts. We identified 116 known genes which were related to diverse functions, with 32.8% relevant for cell cycle or proliferation indicating the potential involvement of these genes in immortalization. We chose eight known genes located on the overrepresented chromosomes of non-small-cell lung cancers (NSCLCs). ASPM, RFC4, C3orf26, BXDC2, C15orf44, AURKA, C20orf77, and RBMX were upregulated in immortalized cells, cancer cells, and non-small-cell lung cancer (NSCLC) tissues. We additionally cloned tow novel genes (CHA-V-97 and CHA-V-165) which showed similar upregulated expression patterns in cells and tissues examined. Identification and further characterization of these genes may provide insights of novel players for immortalization and human carcinogenesis. J. Cell. Biochem. 106: 703-713, 2009. (C) 2009 Wiley-Liss, Inc.