Breast-feeding infants of human immunodeficiency virus (HIV)-infected women ingest large amounts of HIV, but most escape infection. While the factors affecting transmission risk are poorly understood, HIV-specific cytotoxic T-lymphocyte (CTL) responses play a critical role in controlling HIV levels in blood. We therefore investigated the ability of breast milk cells (BMC) from HIV-infected women from the United States and Zambia to respond to HIV-1 peptides in a gamma interferon enzyme-linked immunospot assay. All (n = 11) HIV-infected women had responses to pools of Gag peptide (range, 105 to 1,400 spot-forming cells/million; mean = 718), 8 of 11 reacted to Pol, 7 reacted to Nef, and 2 of 5 reacted to Env. Conversely, of four HIV-negative women, none responded to any of the tested HIV peptide pools. Depletion and tetramer staining studies demonstrated that CD8+ T cells mediated these responses, and a chromium-release assay showed that these BMC were capable of lysing target cells in an HIV-specific manner. These data demonstrate the presence of HIV-specific major histocompatibility complex class I-restricted CD8+ CTLs in breast milk. Their presence suggests a role in limiting transmission and provides a rationale for vaccine strategies to enhance these responses.