Cytotoxic T lymphocytes (CTL) specific for human immunodeficiency virus type 1 (HIV-1) are thought to play an important role in controlling HIV-1 infection. HIV-L-specific CTL are readily demonstrated in unstimulated peripheral blood mononuclear cells (PBMC) of HIV-1nfected adults but less frequently in PBMC from vertically infected children. HIV-1-specific CTL lines were derived from a long-term survivor of vertical HIV-l infection usingPBMC stimulated with a CD3-specific monoclonal antibody and interleukin-2; these lines had Gag- or reverse transcriptase (RT)-specific cytotoxicity. Cytotoxicity was restricted by major histocompatibility complex class I antigen and blocked by antibody to the T cell receptor complex. Fluorescence-activated cell sorting analysis demonstrated their phenotype to beCD3+CD4-CD8+. Unstimulated PBMC from this patient had no detectable HIV-1-specific cytotoxicity when tested against autologous HIV-1 envelope-, Gag-, or RT-expressing target cells. Thus, this child with vertically acquired HIV-1 infection likely has HIV-1-specific CTL precursors despite the absence of circulating, activated HIV-1-specific CTL.