We have observed that human CTL clones lose their specific cytolytic activity and cytokine production under certain stimulation conditions, while retaining an antigen-dependent growth pattern. These inactive CTL simultaneously lose their labeling by an HLA-peptide tetramer, even though the amount of TCR-CD3 at their surface is not reduced. The tetramer-negative cells recover tetramer staining and cytolytic activity after stimulation with tumor cells in the presence of a supernatant of activated lymphocytes. Our results suggest the existence of a new type of functional defect of CTL. They also indicate that tetramers may fail to reveal some CTL bearing the relevant TCR, even when such functionally arrested CTL retain the potential to participate in immune responses because their defect is reversible.