Tetrameric peptide-MHC class I complexes (“tetramers”) are proving invaluable as reagents for characterizing immune responses involving CTLs. However, because the TCR can exhibit a degree of promiscuity for binding peptide-MHC class I ligands, there is potential for cross-reactivity. Recent reports showing that the TCR/peptide-MHC interaction is dramatically dependent upon temperature led us to investigate the effects of incubation temperature on tetramer staining. We find that tetramers rapidly stain CTLs with high intensity at 37 C. We examine the fine specificity of tetramer staining using a well-characterized set of natural epitope variants. Peptide variants that elicit little or no functional cellular response from CTLs can stain these cells at 4 C but not at 37 C when incorporated into tetramers. These results suggest that some studies reporting tetramer incubations at 4 C could detect cross-reactive populations of CTLs with minimal avidity for the tetramer peptide, especially in the tetramer-low population. For identifying specific CTLs among polyclonal cell populations such as PBLs, incubation with tetramers at 37 C improves the staining intensity of specific CTLs, resulting in improved separation of tetramer-high CD8+ cells. Confocal microscopy reveals that tetramers incubated at 37 C can be rapidly internalized by specific CTLs into vesicles that overlap with the early endocytic compartment. This TCR-specific internalization suggests that coupling of tetramers or analogues with toxins, which are activated only after receptor internalization, may create immunotoxins capable of killing CTLs of single specificities.