The adenovirus (Ad) vector is an attractive candidate for vaccines designed to elicit cellular immunity as studies in animals and humans have proven Ad vectors are capable of inducing large transgene-specific T-cell responses. However, given that natural infection by Ad is prevalent globally, pre-existing Ad immunity is a major impediment to the use of recombinant Ad-based vaccines. Though the prevalence of pre-existing neutralizing antibodies has been well characterized, there is a lack of information on the functionality and phenotype of Ad-specific T-cell responses among heterogeneous human cohorts. The lack of protection and possible increased risk of HIV infection in the Merck Ad5 HIV vaccine STEP trial further highlights the need to understand vector-specific immunity in order to produce safe, effective Ad-based vaccines. We aimed to characterize Ad-specific T-cell responses in humans following natural infection and vaccination. Ad-specific T-cell responses were measured by stimulating peripheral blood mononuclear cells (PBMCs) with whole Ad vector or overlapping Ad hexon peptide pools. PBMCs were obtained from 17 healthy adults to study natural infection and longitudinally from 40 participants in Merck phase I Ad5 HIV vaccine studies, 10 of which were enrolled in the STEP trial precursor study using the same vector, dosing, and schedule used for the STEP study. T-cell phenotype and functionality were measured by polychromatic flow cytometry. We found that both CD4+ and CD8+ Ad5-specific T-cells were universally present in subjects independent of their serostatus. Ad5-specific CD8+ T-cells exhibited an effector phenotype and produced the effector functions MIP1α and perforin whereas Ad5-specific CD4+ T-cells had an effector memory phenotype producing IL-2, IFN-γ and TNFα. Ad5-specific T-cells recognized both conserved and variable hexon epitopes making them highly cross-reactive with chimpanzee serotypes. Upon Ad5-based vaccination, Ad5-specific CD4+ T-cells were only transiently expanded and there were no differences in activation or mucosal homing marker expression between Ad5-seronegative and Ad5-seropositive subjects. These data suggest the increased risk of HIV infection observed in the STEP trial was not a result of Ad5-specific CD4+ T-cells. Ad5-specific CD8+ T-cells were also transiently expanded by Ad5-based vaccination, however, there were no changes in functionality. Together, these data suggest though pre-existing Ad-specific T-cells may reduce vaccine efficacy, they should not represent a safety concern for the use of Ad-based vaccines.