When hepatocyte-enriched fractions from neonatal rat livers were cultured for different times in the absence of added growth factors, a population of highly proliferating and migrating fibroblastlike cells appeared. Double immunofluorescence with antibodies to cytokeratin and to vimentin showed a progressive reduction in the number of cytokeratin-positive cells parallel to an increase in the vimentin-positive cells. Some cells with transitional epithelial or migrating morphology coexpressed both intermediate filament proteins. Immunofluorescence with antibodies against hepatocyte differentiation markers showed that shortly after seeding most of the cells were positive to anti-albumin antibodies, but after 1 week in culture, only 10% were positive. Cells presenting albumin and cytokeratin appeared morphologically epithelial. Fibroblastlike cells were not positive for albumin, but some cells with transitional epithelial morphology presented some labels for albumin and for vimentin. Immunofluorescence with antibodies to glutathione-S-transferase subunit Pi and vimentin showed that many fibroblastlike cells were positive for both markers, some of them binucleate. Cultures performed in the presence of dexamethasone, absence of arginine, or on collagen type I matrix had no effect on the behavior of neonatal hepatocytes. The appearance of fibroblastlike cells was ontogenically regulated because the highest increase in the percentage of vimentin-positive cells was observed in cell cultures from livers of 7-and 15-day-old animals. These data provide evidence that neonatal hepatocytes in culture have the potential to dedifferentiate by epithelial-mesenchymal transition and contribute to an understanding of hepatic growth development.