Previous studies have demonstrated that neonatal rat hepatocytes cultured in a serum-free medium adopt a fibroblast-like morphology and form a well-developed vimentin network. By immunoblotting, phase-contrast microscopy, and immunolocalization studies we report here that hepatocyte growth factor (HGF) induces a pronounced migratory/scatter phenotype in neonatal hepatocytes in primary culture. These fibroblast-like cells are highly elongated and adopt a pronounced spindle shape in the presence of this growth factor. Most of them coexpress vimentin and cytokeratin intermediate filaments. Both EGF and TGF-beta1 also induced vimentin expression in cytokeratin-positive cells but this effect was not correlated with a change of epithelial phenotype. Only in DMSO-supplemented cultures was vimentin expression of hepatocytes inhibited and no coexpression was observed in the presence of this factor. Remarkably, the effect induced by HGF was totally inhibited when DMSO, TGF-beta1, or EGF was added to HGF-supplemented cultures. Epithelial sheets of well-defined hepatocytes were observed with these combinations although the complete epithelial morphology was achieved by combining DMSO with a growth factor. Taken together these results suggest that growth and differentiation factors modulate the migratory effect of HGF/SF in hepatocytes.