Abstract: Objective To develop a method for acquisition of efficient and stable retroviral packaging cells on the basis of fluorescence-activated cell sorting (FACS) technique. Methods PA317 cells were transfected by the recombinant retroviral vector pLEGFP via liposome, and the result of transfection was examined using fluorescent microscope. Polyclones and monoclone of the packaging cells were obtained with FACS and identified by PCR and reverse transcriptional PCR (RT-PCR). NIH3T3 cells were used to determine the virus titer in the supernatant. Results Examination under fluorescence microscope confirmed the success of cell transfection with the retrovirus, and polyclonal and monoclonal cells with efficient and stable expression were obtained after FACS. The inserted EGFP gene fragment could be amplified by PCR from the genomic DNA of the polyclonal and monoclonal cells and by RT-PCR from the retrovirus RNA in the supernatant of monoclonal cell culture and some of the monoclonal cell cultures (6/8). Determination of the virus titer in the cell culture supernatant showed efficient viral production by the cells. Conclusion FACS is an efficient method for obtaining stable retroviral packaging cells.