Circulating human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) are seen less frequently in unstimulated peripheral blood mononuclear cells (PBMC) from children with vertically acquired HIV infection than in PBMC from HIV-infected adults. HIV-1 Gag-, reverse transcriptase (RT)-, and envelope (Env)-specific cytotoxic activity was studied in PBMC from HIV-infected children. Only 9% of subjects had Gag- or RT-specific CTL in unstimulated PBMC. However, in PBMC studied after CD3 stimulation, Gag- and Env-specific CTL were found in PBMC from 91% and 78% of HIV-infected children, respectively. Limiting dilution analysis of precursor CTL (pCTL) frequencies in PBMC from children > 12 months old demonstrated Gag- and Env-specific pCTL frequencies from 0.5 to 6.3/10,000 PBMC and from 0.66 to 33.0/10,000 PBMC, respectively. Thus, children with vertically acquired HIV infection have high frequencies of HIV-specific pCTL.