CD8₊ T cells are key effectors of the immune response against human cytomegalovirus (HCMV). A number of HCMV-derived CD8⁺ T cell epitopes are known. Using epitope prediction and subsequent testing for interferon-γ responses by the ELISPOT assay, we identified an optimal human leukocyte antigen (HLA)-A^*0201-restricted CD8⁺ T cell epitope derived from the major immediate early 1 (IE-1) gene product. As many as one-third of HLA-A^*0201-positive, HCMV-seropositive donors make responses to this peptide (residues 316–324 [VLEETSVML]), which can exceed responses against a published immunodominant pp65 epitope (residues 495–503 [NLVPMVATV]). Major histocompatibility complex peptide tetramer staining facilitated detailed phenotypic analyses and revealed populations that resemble terminally differentiated effector cells (CD57⁺ and CD28^-), with considerable restriction in T cell receptor β-chain variable region use. The results confirm that, although pp65 is a major target for CD8⁺ T cells, the IE-1 protein may itself stimulate comparable responses in some persons.