CD8⁺CD28^– T cells are selectively expanded during viral infections, indicating their importance in anti-viral immune responses. Since little is known about the differentiation of CD8⁺CD28^– cells, we investigated the generation, function and survival characteristics of this subset. In healthy individuals CD8⁺CD28^– T cells contained more elevated levels of perforin and IFN-γ than the CD8⁺CD28⁺ subset, indicating that they can have an effector function. CD8⁺CD28^– cells were selectively expanded when activated CD8⁺CD28⁺ T cells were cultured in IL-2, IL-7 or IL-15. Moreover, the generation of CD8⁺CD28^– cells was accelerated by type I IFN suggesting that these cytokines which are released during viral infections influence CD8⁺ T cell differentiation. We did not observe re-expression of CD28 by CD8⁺CD28^– T cells in any of the experiments performed. Activated T cells are susceptible to activation-induced cell death (AICD) if re-stimulated in the absence of co-stimuli. AICD was induced in both CD28⁺ and CD28^– subsets of activated T cells when stimulated with anti-CD3 antibody in the absence of co-stimuli but the magnitude of death was greater in the CD28^– subset. While co-stimulation through LFA-1 (CD11a and CD18) significantly reduced AICD in the CD8⁺CD28⁺ subset, death was not prevented in CD8⁺CD28^– cells. These results suggest that CD8⁺CD28^– T cells are more functionally differentiated than the CD8⁺CD28⁺ subset and indicate they may represent a terminally differentiated effector population which is destined for clearance by apoptosis at the end of the immune response.