Cell-mediated immunity plays an essential role in the control of infection with the human cytomegalovirus (HCMV). However, only a few CD8⁺-T-cell epitopes are known, with the majority being contained in the pp65 phosphoprotein, which is believed to dominate the CD8⁺-T-cell response to HCMV. Here, we have readdressed the issue of CD8⁺ T cells specific for the 72-kDa major immediate-early protein (IE-1), which is nonstructural but is found very early and throughout the replicative cycle. Using a novel flow-cytometric assay, we were able to identify CD8⁺-T-cell epitopes (by IE-1 peptide-specific induction of cytokine synthesis) and simultaneously measure the frequency of cells directed against them. For this purpose, 81 pentadecamer peptides covering the complete 491-amino-acid sequence of IE-1 were tested on peripheral blood mononuclear cells of anti-HCMV immunoglobulin G-seropositive donors. At least 10 new epitopes were identified, and the fine specificity and presenting HLA molecule of the first of them was determined. The frequencies of CD8⁺ T cells directed against IE-1 were similar to those directed against pp65 in donors tested with known pp65-derived peptides. Importantly, additional testing of a corresponding set of peptides covering the complete sequence of pp65 on 10 of these donors identified individuals whose CD8⁺ T cells recognized IE-1 but not pp65 and vice versa, clearly illustrating that either protein may be a major target. In summary, our results suggest that IE-1 is far more important as a CD8⁺-T-cell target than current opinion suggests.