Objective:To establish a reliable method for isolation and culture of murine adult oval cells.Methods :Oval cell response was stimulated by treatment with N2acetylaminofluorene(2AAF) diet and partial hepatectomy. A modification of a twostep perfusion protocol and isopycnic centrifugation were used to isolate oval cells from livers. Immunofluorescence and doublefluorescence immunostaining were used for identification of oval cells.Results:Isolated oval cells were grown as colonies in vitro and had been cultured for three months. Isolated cells were identification by immunofluorescence and doublefluorescence immunostaining, which also indicated isolated cells were bipotential.Conclusions:This method mentioned above is a reliable method for isolation and primary culture of oval cells, which would provide a useful tool for investigating oval cell biology.