The loading of MHC class I molecules with their peptide cargo is undertaken by a multimolecular peptide loading complex within the endoplasmic reticulum. We show that MHC class I molecules can optimize their peptide repertoire over time and that this process is dependent on tapasin. Optimization of the peptide repertoire is both quantitatively and qualitatively improved by tapasin. The extent of optimization is maximal when MHC class I molecules are allowed to load within the fully assembled peptide loading complex. Finally, we identify a single natural polymorphism (116D>Y) in HLA-B*4402 that permits tapasin-independent loading of HLA-B*4405 (116Y). In the presence of tapasin, the tapasin-independent allele B*4405 (116Y) acquires a repertoire of peptides that is less optimal than the tapasin-dependent allele B*4402 (116D).