The effectiveness of peptide-based cancer vaccines depends on the ability of peptides to bind to MHC molecules on the surface of antigen-presenting cells, where they reconstitute epitopes for cytotoxic T lymphocytes (CTLs). Multivalent vaccines have advantages over single-peptide vaccines; however, peptides may compete for binding to the same MHC molecules. In particular, it is possible that peptides with high affinity for MHC molecules prevent the binding of lower-affinity peptides. However, only small numbers of peptide/MHC complexes per cell are required for CTL recognition. Thus, the authors hypothesized that competition of peptides for MHC binding would not significantly reduce CTL recognition of individual peptides within a multiple-peptide mixture, and this hypothesis was tested by a series of experiments performed in vitro. In multiple experiments, two peptides with different affinities for HLA-A*0201 molecules were mixed at various concentrations and pulsed onto HLA-A2 cells, which were then evaluated for susceptibility to lysis by HLA-A*0201-restricted CTLs. CTL recognition of the melanoma peptides gp100(154-162) (KTWGQYWQV), gp100(280-288) (YLEPGPVTA), and tyrosinase(369-377D) (YMDGTMSQV) was maintained even when target cells were co-pulsed with equimolar concentrations of peptides with comparable or higher affinity for HLA-A2. In some cases, CTL recognition was maintained even when the higher-affinity peptide was present at concentrations several orders of magnitude higher than the target peptide. In addition, CTLs generated by in vitro stimulation with a peptide mixture developed reactivity to three different peptides, at a level comparable to that obtained by stimulation with each individual peptide separately. These data suggest that CTLs can respond to multiple peptides presented on the same antigen-presenting cells and justify further investigation, in clinical trials, of multiple-peptide cancer vaccines.