The generation of memory lymphocytes is one of the hallmarks of the specific immune response. The CD4+ T cell response is of critical importance in maintaining long-term protective immunity after clearing many infections. However, accurate characterization of these memory CD4+ T cells has relied mainly on mouse studies and is poorly understood in humans. We have detected and counted epitope-specific populations of CD4+ memory cells in patients who have cleared hepatitis C virus. The kinetics of the recall response and the expression of the chemokine receptor CCR7 suggested the presence of distinct populations. A population of memory cells measured in an ex vivo IFN-γ ELISPOT assay steadily declined after viral clearance. However, memory CD4+ T cells only characterized after short-term culture with Ag and IL-2, and, recognizing the same epitopes, developed into a long-term stable population. Depletion of CCR7+ cells from PBMCs markedly reduced the responses in the culture-positive population while having little effect on the ex vivo responses. The demonstration of these key memory subsets in man opens the way to defining their role in protective immune responses.