The control of many persistent viral infections by Ag-specific cytolytic CD8⁺ T cells requires a concurrent virus-specific CD4⁺ Th cell response. This reflects in part a requirement of activated effector CD8⁺ T cells for paracrine IL-2 production as a growth and survival factor. In human CMV and HIV infection, the majority of differentiated virus-specific CD8⁺ T cells notably lose the ability to produce IL-2 but also lose expression of CD28, a costimulatory molecule. Analysis of the fraction of memory CD8⁺ T cells that continue to express CD28 revealed these cells retain the ability to produce IL-2. Therefore, we examined if IL-2 production by CD28⁻ CD8⁺ T cells could be restored by introduction of a constitutively expressed CD28 gene. Expression of CD28 in CD28⁻ CD8⁺ CMV- and HIV-specific CD8⁺ T cells reconstituted the ability to produce IL-2, which could sustain an autocrine proliferative response after Ag recognition. These results suggest that the loss of CD28 expression during differentiation of memory/effector CD8⁺ T cells represents a decisive step in establishing regulation of responding CD8⁺ T cells, increasing the dependence on CD4⁺ Th for proliferation after target recognition, and has implications for the treatment of viral disease with adoptively transferred CD8⁺ T cells.