We have recently reported that cell lines of nonparenchymal origin isolated from rat liver and pancreas, which have been suggested to be the progeny of a facultative stem cell compartment in vivo, express an unusual combination of keratins (K). These cell lines express K8 and K14 but not K18 and K5, their normal partners in filament formation (Bisgaard HC, Thorgeirsson SS, J Cell Physiol 147:333-343, 1991). However, upon spontaneous transformation and differentiation toward a hepatoblastlike progeny, K14 expression is abrogated and replaced by expression of K18 (Wirth et al., Electrophoresis 13:305-332, 1992). In the study presented here, we confirmed by protein sequence analysis that K14 was a major component of the intermediate filaments in a nonparenchymal cell line of hepatic origin. Immunocytochemical analysis of the cells in monolayer demonstrated that K8 as well as K14 were incorporated in the cellular cytoskeleton. Further analysis by immunoprecipitation showed that filament complexes were formed between K8 and K14 as atypical partners. Thus, we concluded that in some nonparenchymal cell lines isolated from rat liver, K8 and K14 form a major intermediate filament network. Finally, we showed that an antibody widely used in studies of the cell lineages of hepatic and pancreatic tissues and their neoplasms, the mouse monoclonal antibody OV-6, recognizes a common epitope in K14 and K19.