Adenoviruses (Ads) cause fatal disease in allogeneic stem cell transplant recipients, but there is no established therapy. Ad-specific CD8⁺ T cells were detected in PBMC from healthy adults at a mean frequency of 77 per 10⁵ CD8⁺ T cells (range 8–260) by interferon-γ ELISPOT and cytokine flow cytometry assays. CD8⁺ T cell lines from 7 of 7 donors exhibited MHC-class-I-restricted killing of targets expressing the capsid protein hexon. In contrast, cytotoxicity against the capsid proteins fiber and penton base was weaker or not detected. Two HLA-A2-restricted hexon epitopes and one HLA-B-restricted epitope were identified, all of which are adjacent to or overlap an HLA-DP4-restricted epitope in the highly conserved C-terminus. Thus, hexon is the immunodominant T cell target among capsid proteins and contains multiple C-terminal epitopes conserved among serotypes. These data support evaluation of donor lymphocyte infusions for treatment of Ad disease post-transplant.