Apoptosis of CD4+ and CD8+ T cells has been shown in peripheral blood mononuclear cells (PBMCs) from HIV-infected adults analyzed after overnight culture. Because cell death may be an artifact of in vitro culture, and because there is little information on apoptosis in pediatric HIV disease, we undertook a cross-sectional analysis of apoptosis in PBMCs analyzed immediately ex vivo in HIV-infected children and adults. PBMCs from 22 children, four adolescents, and nine adults and seronegative age-matched control subjects were stained for CD4 and CD8 surface markers. Apoptotic cells were detected in a newly characterized flow cytometric assay by diminished forward and increased side scatter. Children with the most advanced disease had 9.9% (SEM 1.8) apoptotic CD4+ T cells above control, significantly higher than in asymptomatic patients [0.4% (SEM 2.3)], those with mild disease [2.2% (SEM 1.83)], and those with moderate disease [2.5 (SEM 3.6)] (p = 0.015). The percentages of both CD4+ and CD8+ T cell apoptosis were directly related to CD4+ T cell depletion (R2 = 0.23; p = 0.006; n = 32 and R2 = 0.2; p = 0.012; n = 30, respectively). Patients who responded to antiretroviral therapy with the greatest increase in CD4+ T cell percentage had the least CD4+ T cell apoptosis (R2 = 0.15; p = 0.1; n = 19). These findings show that the rate or extent of T cell death by apoptosis percentage of T cell apoptosis is significantly increased in HIV-infected children. The observed correlation of both CD4+ and CD8+ T cell apoptosis with CD4+ T cell depletion suggests that apoptosis plays a role in HIV pathogenesis and may be a useful marker of disease activity.