Detection of the functional CD8⁺ CTL response usually requires in vitro restimulation. The differences between the CD8⁺ CTL repertoire in freshly isolated precursor cells and CD8⁺ CTL after short‐term in vitro expansion have been generally assumed to be minimal, but have never been defined experimentally. Using staining with P18‐I10/H‐2D^d tetramers and monoclonal antibodies (mAb) against Vβ, we show the surprising result that there was significant skewing of the CD8⁺ CTL repertoire after just 7 days of stimulation. In contrast, we found that overnight incubation of precursor cells with peptide allows the functional assessment of CD8⁺ CTL (which cannot be detected ex vivo from freshly isolatedcells) without changing the absolute number of antigen‐specific CTL as measured by tetramer staining or the repertoire of TCR analyzed with mAb. This study affords a better understanding of the differences between the ex vivo and in vitro stimulated CTL repertoire, and provides an approach to reveal a more faithful representation of the functional in vivo CTL response without skewing of the repertoire of T cells detected.