To probe the mechanism of carcinogenesis of lung cancer at the molecular level and to find potential protein markers involved in the early phase of tumorgenesis, differential proteome analysis on primary passage cell line R15H, and early transformed cell line R15H20 derived from (238)Pu alpha-particle irradiation of human papillomavirus (HPV) 18-immortalized human bronchial epithelial cell line (BEP2D), was carried out using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Image analysis and Student's t-test (p < 0.05) showed that three protein spots were only expressed in R15H, intensities of 43 protein spots on the gels were altered between R15H and R15H20. Two of the three spots that were only expressed in R15H were identified as high mobility group protein 1. Two proteins decreased in abundance in R15H20 were identified as maspin precursor, a tumor suppressor and aminoacylase-1. Ornithine aminotransferase and peptidyl-prolyl cis-trans isomerase A that were increased in R15H20, were also identified. Relationships between these differentially expressed proteins and the carcinogenesis mechanism of lung cancer are discussed. The protein expression profile of the R15H cell line was also constructed during the study as a reference map for further comparative proteome analysis of the irradiation induced BEP2D cell line. Of the 90 spots analyzed with PMF in the 2-DE gel of R15H cell line, 50 proteins were identified by searching the nonredundant protein database SWISS-PROT/TrEMBL.