To search for a reliable biomarker of preneoplastic lesions arising early in mouse hepatocarcinogenesis the proteomes of microdissected basophilic foci, hepatocellular adenomas (HCAs), carcinomas (HCCs) and normal-appearing liver of B6C3F1 mice initiated with diethylnitrosamine (DEN) were analysed on anionic (Q10) surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip arrays. Significant overexpression of cytokeratin 8 (CK8; m/z 54, 565), cytokeratin 18 (CK18; m/z 47,538) proteins was found in basophilic foci as well as in HCAs and HCCs. Furthermore, immunohistochemistry demonstrated profound overexpression of CK8 and CK18 proteins (CK8/18) in all basophilic foci, mixed cell type foci, HCAs and HCCs in B6C3F1 and C57BL/6J mice initiated with DEN. A strong correlation between CK8/18-positive foci development and multiplicity of liver tumors in B6C3F1 and C57Bl/6J mice was further observed. Moreover, formation of CK8 and CK18 complexes due to CK8 phosphorylation at Ser73 and Ser431 was found to be strongly associated with neoplastic transformation of mice liver basophilic foci. Elevation of CK8/18 was strongly correlated with induction of cell proliferation in basophilic foci and tumors. In conclusion, our data imply that CK8/18 is a novel reliable marker of preneoplastic lesions arising during mouse hepatocarcinogenesis which might be used for prediction of tumor development and evaluation of environmental agents as well as drugs and food additives using mouse liver tests.