Adoptive immunotherapy with virus-specific T lymphocytes can efficiently reconstitute antiviral immunity against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (ADV) without causing acute toxicity or increasing the risk of graft-versus-host disease. To gain insight into antiviral T cell repertoires and to identify the most efficient antigens for immunotherapy, the frequencies of CMV-, EBV- and ADV-specific T cells in 204 HLA-typed healthy donors were assessed using viral peptides and peptide pools. Confirmatory testing for CMV serology by Western blot technique revealed 19 of 143 (13%) false-positive results. We observed highly significant individual and overall differences in T cell frequencies against CMV, EBV, and ADV antigens, whereas antigen-specific T cells were detected in 100% of CMV- seropositive donors, 73% of EBV- seropositive donors, and 73% of ADV-seropositive donors. At least 124 (61%) potential T cell donors were identified for each virus. Among the tested antigens, frequencies for CMVpp65 and EBVBZLF1 peptide pools were highest. Short-term in vitro peptide stimulation revealed that a donor response to a certain ADV- and EBV-derived peptide may not be determined without prior stimulation. A modified granzyme B ELISpot was used to detect T cell specificity and alloreactivity. Treatment with allogeneic virus-specific cytotoxic T lymphocytes from seropositive third-party donors may be a feasible therapeutic option for infections following cord-blood stem cell transplantation or hematopoietic stem cell transplantation from virus-seronegative donors.