A reproducible, semiquantitative assay was developed to measure the level of the anti-human immunodeficiency virus type 1 (anti-HIV-1) suppressive activity of CD8+ T cells. The assay had a wide dynamic range and could be applied to a relatively small number of fresh and cryopreserved peripheral blood mononuclear cells and purified CD8+ T cells. The suppressive activity was not due to cytolytic activity and was not major histocompatibility complex class I restricted. Suppression of HIV-1 infection by CD8+ T cells was consistently demonstrable with both endogenously infected autologous CD4+ T cells and exogenously infected allogeneic CD4+ T cells. This assay can be used to monitor the level of antiviral activity of CD8+ T cells in a retrospective and prospective manner in studies of the natural history of HIV-1 infection and of subjects receiving anti-HIV-1 therapy and vaccines.