To investigate the effect of highly active antiretroviral therapy (HAART) on HIV‐ and Epstein‐Barr virus (EBV)‐specific CD8⁺ T cells, total number and function of these cells was determined in 16 HIV‐infected individuals using tetrameric HLA‐peptide complexes and IFN‐γ ELISPOT assays after peptide stimulation, respectively. HAART induced a significant decrease in HIV‐specific tetramer⁺ T cells, whereas EBV‐specific tetramer⁺ T cells did not change. In addition, individuals who temporarily failed on therapy showed a temporary increase in the number of HIV‐specific T cells, suggesting that differences in the pool size of antigen‐specific T cells was determined by the presence of antigen. Interestingly, there was an increase in the ratio of IFNγ‐producing T cells per total number of both HIV‐ and EBV‐specific T cells in the majority of individuals, suggesting that the function of virus‐specific T cells is improved in individuals successfully treated with HAART. Despite this relative functional improvement of EBV‐specific T cells, no significant changes were observed in EBV load. In four subjects who temporarily failed on HAART, the percentage of IFN‐γ‐producing T cells, both for HIV and EBV, paralleled CD4⁺ T cell kinetics, suggesting that function seems to be related to differences in CD4⁺ T cell numbers. Overall, these data indicate that HAART improves the antigen responsiveness of both HIV‐ and EBV‐specific T cells, which is associated with an increase in CD4⁺ T cells.