Individuals infected with human immunodeficiency virus (HIV) have low numbers of functional Epstein-Barr virus (EBV)-specific CD8+ T cells in the face of a high EBV load, suggesting that these cells have become exhausted. We investigated whether the observed chronic EBV loads during HIV infection could cause exhaustion of EBV-specific T cells by using flow-FISH (flow cytometry in combination with fluorescence in situ hybridization) to analyze the telomere length of EBV-specific CD8+ T cells. Enhanced telomere shortening of EBV-specific T cells was observed during HIV infection, compared with the decline in telomere length observed in the CD8+ T cells of healthy subjects. Thus, chronic exposure to high antigen levels may lead to the progressive shortening of telomeres of antigen-specific T cells, which may impair viral control.