CD8+ T cells provide protective immune responses via both cytolytic and non-cytolytic mechanisms in subjects infected with human immunodeficiency virus (HIV). In the present study, we investigated the CD28 expression of CD8+ T cells present in the peripheral blood lymphocyte subset isolated from chronically HIV-infected subjects. Using flow cytometric analysis, a continuous spectrum of CD28 intensity ranging from negative to high, which could be separated into CD28-negative, intermediate (int) and high, was seen for CD8+ T cells. Our study focused mostly on the CD28int CD8+ T cells. The CD28int CD8+ T cells are CD57– CD27+ CD45RO+ CD45RA– CCR7low CD62Lint. The proliferative capacity of CD28int CD8+ T cells was intermediate between those of CD28– CD8+ T cells and CD28high CD8+ T cells. The CD28int CD8+ T cells are specific for HIV, cytomegalovirus (CMV) and Epstein–Barr virus (EBV) antigens as measured by human leucocyte antigen pentamer binding and produce both intracellular interferon-γ and tumour necrosis factor-α in response to their cognate viral peptides. The CD28int CD8+ T cells have HIV-specific, CMV-specific and EBV-specific cytotoxic activity in response to their cognate viral peptides. These findings indicate that a subset of functional effector-memory CD8+ T cells specific for HIV, CMV and EBV antigens may contribute to an efficient immune response in HIV-infected subjects.