The induction of CD8+ CTL responses is a goal of most HIV-1 vaccine trials, but such potentially protective effector responses have been difficult to evaluate, particularly in these vaccine prevention trials, due to technical obstacles. We report a method to evaluate CTL responses based on the ability to infect autologous macrophages with a monocytotropic strain of HIV-1, and to use these cells as efficient stimulators. This approach does not require the addition of exogenous cytokines, allows detection of class I-restricted CTLs against multiple HIV-1 gene products, and circumvents the problem, often detected using other stimulator cells, of high levels of lytic activity against target cells expressing vaccinia and/or EBV antigens. Adherent monocyte-derived macrophages were infected with HIV-1 Ba-L, and used within 2-3 weeks as autologous stimulators. Fresh PBMCs were cultured with the infected macrophages, harvested after 1 week, replated with fresh infected macrophages and filler cells, and tested after 5-7 days for cytolytic activity. CD8+ CTL responses specific for HIV-1 envelope were detected at an E:T ratio as low as 5:1 in two of four HIV-1-uninfected recipients of an HIV vaccine regimen that included a recombinant live vaccinia virus. Cytotoxic T lymphocyte activity could be detected > 1 year following vaccination. Similar lytic activity was detected with cryopreserved responder cells. In two HIV-1-infected individuals participating in a blinded therapeutic vaccination trial, the use of infected macrophages as in vitro stimulators permitted detection of the presence of envelope and gag-specific CTLs. No responses were observed in nonimmunized, uninfected controls. Thus, HIV-1-infected macrophages can stimulate in vitro the repertoire of primed HIV-reactive CD8+ precursors from seronegative and seropositive participants in HIV-1 vaccine trials, and should facilitate the identification of potentially effective candidate HIV vaccines.