Cytotoxic T lymphocytes (CTL) that kill uninfected activated CD4+ T cells can be induced in vitro by stimulating CD8+ T cells with activated autologous CD4+ T cells. Similar CTL have been detected in circulating T cells from human immunodeficiency virus type I (HIV)-infected individuals. To define the in vivo correlates of this CTL activity, we studied plasma beta-2 microglobulin and HIV RNA levels, T-lymphocyte subset counts, and expression of CD28 on CD8+ T cells concurrently with circulating CTL activity against uninfected CD4+ T cells in 75 HIV-infected individuals at different stages of disease progression. Mean values of each parameter were compared in subsets of this group of 75 segregated on the basis of this CTL activity. The group with CTL against uninfected activated CD4+ T lymphocytes had more CD8+ T cells, a higher percentage of CD28 CD8+ T cells, and higher plasma levels of HIV RNA and beta-2 microglobulin. CTL against uninfected activated CD4+ T cells were predominantly CD28 and in HIV-infected individuals were associated with immunological or virological evidence of progressive disease. In HIV infection, circulating CTL activity against uninfected activated CD4+ T lymphocytes is associated with immune activation, CD8+ T cell expansion, accumulation of CD28 CD8+ T cells, and inadequate suppression of HIV replication.