We have shown that subcultured keratinocytes derived from normal human skin and grown on plastic in serum-free defined medium (MCDB 153, with 0.1 mM Ca2+ concentration) synthesize vimentin. A fibrillar vimentin expression was observed in the cell cytoplasm by immunohistochemistry with different monoclonal antibodies to vimentin in a high proportion of cultured cells. Two dimensional gel electrophoresis of cytoskeletal proteins from keratinocyte subcultures and immunoblotting reaction with different monoclonal antibodies to vimentin showed a specific reaction in a position corresponding to vimentin. No cross-reaction with keratin polypeptides was obtained. This expression of vimentin may be related to the adaptation of cells to in vitro growth conditions.