The Env glycoprotein of human immunodeficiency virus is critical for the pathogenesis of the acquired immunodeficiency syndrome, and has been the prime target for candidate HIV-1 vaccines. Cytolytic T lymphocytes (CTLs) may be important for the immunologic control of HIV infection and HIV-1 Env-specific cytolytic T cells have been isolated from infected individuals and seronegative recipients of HIV-1 vaccines. Most prior studies have used assays that detect Env-specific CTLs directed against standard laboratory viral variants. These studies may be limited because the Env proteins of these laboratory strains (for example, LAI and MN) may differ significantly from the Env proteins from primary HIV-1 strains, and a single amino acid change can abrogate the recognition of HIV-1 Env by some CTL clones. Therefore, this study measured CTL activity directed against HIV-1 Env representing the infected individual's (autologous) HIV-1 viral variants. For two HIV-1-infected individuals, recombinant vaccina viruses expressing cloned HIV-1 env genes were constructed. Using an in vitro stimulation method, strain-specific CTL activity directed against autologous HIV-1 Env was detected in both individuals. From one subject, strain-specific CTL clones directed against autologous and HIV-1LAI Env were characterized. Therefore, some infected individuals have Env-specific CTLs directed against autologous strains of HIV-1. Detection and characterization of autologous Env-specific CTL activity may have important implications relative to the current HIV-1 vaccine development strategies focusing on Env derived from laboratory strains of HIV-1.