Although an increased frequency of CD4⁺CD8⁺ T cells has been observed in the peripheral blood during viral infections, their role, function, and biologic significance are still poorly understood. Here we demonstrate that the circulating CD4⁺CD8⁺ T-cell population contains mature effector memory lymphocytes specific for antigens of multiple past, latent, and high-level persistent viral infections. Upon in vitro antigenic challenge, a higher frequency of CD4⁺CD8⁺ than single-positive cells displayed a T helper 1/T cytotoxic 1 (Th1/Tc1) cytokine profile and proliferated. Ex vivo, more double-positive than single-positive cells exhibited a differentiated phenotype. Accordingly, their lower T-cell receptor excision circles (TREC) content and shorter telomeres proved they had divided more frequently than single-positive cells. Consistent with expression of the tissue-homing marker CXCR3, CD4⁺CD8⁺ T cells were demonstrated in situ at the site of persistent viral infection (ie, in the liver during chronic hepatitis C). Finally, a prospective analysis of hepatitis C virus (HCV) infection in a chimpanzee, the only animal model for HCV infection, showed a close correlation between the frequency of activated CD4⁺CD8⁺ T cells and viral kinetics. Collectively, these findings demonstrate that peripheral CD4⁺CD8⁺ T cells take part in the adaptive immune response against infectious pathogens and broaden the perception of the T-cell populations involved in antiviral immune responses. (Blood. 2004;104:478-486)