Human immunodeficiency virus type 1 (HIV-1)–infected individuals, but not HIV-seronegative controls, have non–HLA-restricted T-cell receptor αβ+CD8+cytotoxic T-lymphocytes (CTL) that kill activated uninfected CD4+lymphocytes. In vitro stimulation of peripheral blood mononuclear cells from HIV-1-infected individuals with concanavalin A (Con A) or by coculture with phytohemagglutinin-activated autologous lymphoblasts induced CTL that killed autologous and heterologous CD4+lymphocytes, but not Con A–activated CD8+lymphocytes or Epstein-Barr virus (EBV)–transformed B lymphocytes. EBV did not stimulate such CTL in two subjects tested, although stimulation with Con A or autologous lymphoblasts induced CTL activity against CD4+lymphocytes in both subjects. CTL activity against autologous CD4+lymphocytes varied over time; killing of heterologous CD4+lymphocytes was often higher than that of autologous CD4+lymphocytes. HIV-infected individuals with Con A–inducible CTL against autologous CD4+lymphocytes lost more CD4+lymphocytes within 6 months of testing than HIV-infected individuals with no such CTL (p < .01). The mean (±SD) decrease in CD4+lymphocyte counts in a group of HIV-infected individuals with CTL activity against autologous CD4+lymphocytes was 121 ± 84, or 36%, of total CD4+lymphocytes over 6 months. In contrast, there was no significant change in mean CD4+lymphocyte count over 6 months in a group of HIV-infected individuals without CTL activity against autologous CD4+lymphocytes. In some HIV-infected individuals. CTL activity against autologous CD4+lymphocytes fell coincident with a drop in CD4+lymphocyte number in vivo. The same individuals maintained CTL activity against CD4+lymphocytes from an uninfected individual, consistent with elimination of autologous CD4+targets of CTL from the HIV-infected individuals in vivo. These results suggest that CTL against CD4+lymphocytes contribute to CD4+lymphocyte loss in HIV infection.