Updated project metadata. In healthy individuals, immune control of persistent human cytomegalovirus (HCMV) infection is effectively mediated by virus-specific CD4+ and CD8+ T cells. However, identification of the repertoire of T-cell specificities for HCMV is hampered by the immense protein coding capacity of this betaherpesvirus. We applied a novel approach which employs HCMV deletion mutant viruses, lacking HLA class I immunoevasins. Infection of human fibroblast cell lines with those mutant viruses allowed the direct identification of naturally presented HCMV-derived HLA ligands by mass spectrometry. Using this strategy, we identified 368 unique HCMV-derived HLA class I ligands representing an unexpectedly broad panel of 123 HCMV antigens. Functional characterization revealed memory T-cell responses in seropositive individuals for a substantial proportion (28%) of these novel peptides. The unbiased identification of naturally presented viral epitopes enabled a comprehensive and systematic assessment of the physiological repertoire of anti-HCMV T-cell specificities in seropositive individuals. This approach proved to be superior to procedures applying in silico analysis to identify true viral antigens.