Adenovirus (Ad) infection is a cause of significant morbidity and mortality in hematopoietic stem cell transplant recipients and virus-specific immunotherapy is one option for improved control. Cellular immunity is an important component in suppression of Ad replication but the frequency and population distribution of Ad-specific CD8 T cells has not been systematically investigated. This is an important question in relation to the potential use of these cells for adoptive transfer. To address this question, HLA-peptide multimers were generated for 8 HLA class I-restricted Ad epitopes, which are highly conserved across Ad species. Epitope-specific CD8 T cells from healthy donors were identified by tetramer staining and HLA class I A*01-restricted TDL peptide staining T cells were characterized in relation to frequency, phenotype, and function. The cells demonstrated a minimally differentiated central memory phenotype (CD45RA, CD45RO, CCR7, CD62L, CD27, CD28, and CD57) and were able to produce IFN-γ and proliferate extensively upon antigen stimulation in vitro. After proliferation, the phenotype switched to CD45RO, although it is interesting to note that CCR7 expression was retained. Despite their low frequency, tetramer-staining cells could be enriched with magnetic bead technology. Their characteristics should permit rapid establishment in vivo post adoptive transfer, increasing therapeutic options for patients with Ad infection. This is the first reported characterization of Ad-specific tetramer-staining T cells with a view to adoptive transfer to hematopoietic stem cell transplant patients with Ad infection. The efficacy of these cells needs to be further evaluated in the setting of a clinical trial.