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Abstract. Rat liver epithelial cells (LECs) are non-parenchymal proliferating cells that readily emerge in primary culture and can be established as cell lines, but their in vivo cell(s) of origin is unclear. We reported recently some evidence indicating that the LEC line, T51B, contains two cytokeratins (CKs) equivalent to human CK8 and CK14 respectively. T51B cells also contain vimentin assembled as a network of intermediate filaments distinct from that of the CKs. In the present study, we examined the expression of CK14 gene in various LEC preparations and a Triton-resistant rat skin cytoskeletal fraction, and then assessed its usefulness as an LEC specific marker in the liver. Northern and Western blot analyses with cDNAs and antibodies for CK8, CK14, CK18 and vimentin confirmed that rat hepatocytes express CK8 and CK18 genes only, whereas T51B cells express CK8, CK14 and vimentin genes in the absence of CK18. CK14 was also present in LECs derived as primary from embryonic-day 12 rat liver and secondary cultures from 4-day-old rat liver. Primary cultures of oval cells isolated from 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) treated rat liver (an enriched source of biliary epithelial cells) contained CK14 mRNAs which were slightly shorter than those in LECs. The analyses of CK5 (the usual partner of CK14) gene expression using specific cDNA and antibody clearly demonstrated its absence in LECs. In situ double immunolocalization analyses by laser scanning confocal microscopy showed that CK14 was not present in hepatocytes (HES+6 cells) and was expressed in some biliary epithelial (BDS7+ cells). CK14-positive cells were also found in the Glisson's capsule. However, CK14-positive cells of the portal region were vimentin negative, whereas those of the Glisson's capsule were vimentin positive. Our results suggest that CK14 gene expression is part of the differentiation program of two types of LECs and that this differential CK14 gene expression can be used as a new means to type LECs in culture and in vivo.