The ability to induce a strong, HIV-1-specific CD8+ CTL response is assumed to be an important component of a protective HIV-1 vaccine. Identification of CTL responses in seronegative vaccines requires in vitro stimulation of CTL precursors with cells that have processed HIV-1 gene products via the endogenous intracellular pathway for presentation in association with MHC class I molecules. We have developed a method to detect CTL responses to HIV epitopes and HIV-infected cells that can be applied to recipients of HIV vaccines regardless of immunization with a particular recombinant virus or prior immunological status. Primed CTL precursors from PBMCs are stimulated for two 1-week cycles with autologous monocyte-derived macrophages infected with HIV-1Ba-L. The effector CTLs generated in culture are then tested in a standard chromium release assay for lysis, using autologous target cells, including EBV-transformed lymphoblastoid cell lines (LCLs) expressing individual HIV-1 proteins following infection with a recombinant vaccinia virus, or LCLs transduced with the CD4 gene and infected with isolates of HIV. Using this methodology we have examined CTL responses induced by candidate HIV vaccines in HIV-1-uninfected individuals participating in phase I/II AVEG trials. Our findings indicate that this approach makes it possible to overcome some of the previous technical difficulties involved in the analysis of CTL responses in immunocompetent vaccine recipients and thereby facilitates the identification of potentially effective candidate HIV-1 vaccines.