Although highly active antiretroviral therapy against human immunodeficiency virus (HIV) type 1 reduces the mortality of persons with acquired immunodeficiency syndrome, it does not eliminate HIV reservoirs. In this study, which used a 6-thioguanine (6-TG) resistant clone (4C6) of the MT-2 cell line as a model, the combination of 6-TG with both reverse-transcriptase (RT) inhibitor and protease inhibitor or 6-TG with a protease inhibitor alone completely eradicated HIV-1-carrying cells from the culture and protected uninfected 4C6 cells from HIV-1 infection. The combination of 6-TG and a RT inhibitor, azidothymidine, provided partial protection. Protection was extended to human peripheral blood mononuclear cells. These results suggest that adding a cytotoxic drug in combination antiviral chemotherapy may reduce the establishment of virus reservoirs and prevent virus spread. The clinical value of this and similar strategies should be further evaluated in HIV-infected patients.