A human cell line constitutively expressing the HIV-1 gag and pol genes products was established. The cell line was established by stably transfecting 293 cells with a plasmid construct that expresses the HIV Gag and Pol and can confer the transfectants resistant to mycophenolic acid. Particles generated from transient expression of the plasmid construct were noninfectious when pseudotyped with HIV envelope or with amphotropic murine leukemia virus envelope proteins. However, virus-like Gag particles produced by the stable cell line were appropriately processed, exhibited a wild-type retrovirus particle density, and possessed significant reverse-transcriptase (RT) activities. Continuous passage of the cell line either in the presence or absence of mycophenolic acid had no major effects on the Gag processing efficiency, particle assembly, or RT activity release. It was also demonstrated that the proteolytic processing of the virus-like particles released from the cell line was inhibited by an HIV protease inhibitor, saquinavir. The establishment of a stable cell line producing noninfectious but proteolytically processed HIV Gag particles offers a safe, convenient tool for biochemical and immunological analysis of virus-like particle assembly and is very useful for the development of anti-HIV protease drugs.