The role of human cytomegalovirus (HCMV)–specific T-cell responses in breast milk of HCMV-seropositive mothers is not well defined. In these studies, we demonstrate that the frequency of cytomegalovirus (CMV)-pp65–specific T-cell responses in peripheral blood mononuclear cells (PBMCs) and breast milk cells (BMCs) is increased for CD8⁺ T cells in both sample sources when compared with CD4⁺ T cells. The frequency of pp55-specific CD8 T cells producing interferon γ (IFN-γ) alone or dual IFN-γ/granzyme rB producers is increased in breast milk compared with PBMCs. Lastly, we observed a positive correlation between breast milk viral load and the CD8 pp65-specific response, suggesting that local virus replication drives antigen-specific CD8 T cells into the breast.