CD8 T lymphocytes are an important component of the host immune response to human immunodeficiency virus (HIV). To characterize CD8 cell function, we have studied the in vitro phenomenon of CD8 cell-mediated inhibition of HIV replication from autologous, naturally infected CD4 cells. We describe here a reproducible assay of CD8 T cell-mediated inhibition in HIV-infected individuals. The method involves the use of a commercially available cell separation system and anti-CD3 monoclonal antibody to stimulate CD4 cells to produce HIV. Using this technique, we were able to detect HIV production from the CD4 cells of 25 of 27 HIV-infected individuals who had not progressed to AIDS. Further, in vitro CD8 cell-mediated inhibition of HIV production was noted in all of the 25 subjects whose CD4 cells produced viral p24 antigen. This assay may be useful as an in vitro correlate of protective immunity to HIV, with potential application for assessing disease progression, therapeutic efficacy, and immune mechanisms in HIV disease.