Adoptive transfer of donor-derived cytomegalovirus (CMV)-specific T cells may provide long-lived protection from CMV disease after allogeneic stem cell transplantation. Isolation of IFNg-secreting cells after CMV peptide stimulation can be performed by IFNg capture assay to generate highly specific T-cell lines without the need for extensive culture, which may hamper their in vivo efficacy. To exploit the full potential of this approach, we analyzed the IFNg response of CMV-specific CD8+ T cells in detail. Kinetic studies showed that T-cell receptor down-regulation coincided with the induction of IFNg production upon activation, which rapidly declined thereafter despite the continued presence of specific peptide. By varying the strength of stimulation we observed that overstimulation can result in profound T-cell receptor down-regulation, more rapid decline of IFNg production and reduced expansion. On the basis of these findings, we defined optimal conditions for IFNg-based isolation of CMV-specific CD8+ T cells with maximal potential for clinical application. These data stress the importance of analyses of the kinetics of cytokine production for isolation of T cells specific for other infectious or malignant antigens to exploit the full potential of cytokine capture isolation of antigen-specific T cells.