Understanding how peptides are selected for presentation by MHC class I is crucial to vaccination strategies based on cytotoxic T lymphocyte priming. We have studied this selection of the MHC class I peptide repertoire in terms of the presentation of a series of individual peptides with a wide range of binding to MHC class I. This series was expressed as minigenes, and the presentation of each peptide variant was determined with the same MHC class I peptide-specific antibody. In wild-type cells, the hierarchy of presentation followed peptide half-life. This hierarchy broke down in cells lacking tapasin but not in cells lacking calreticulin or in cells lacking transporter associated with antigen processing-associated ERp57. We demonstrate a key role for tapasin in shaping the MHC class I peptide repertoire, as enhancement of presentation in the presence of tapasin correlated with peptide half-life.