Functional polymorphism in the rat peptide transporter associated with antigen processing (TAP) changes the peptide pool available for binding and presentation by a class I MHC allele, RT1.A^a. The peptide binding motif for RT1.A^a, determined by stabilization with synthetic peptides, included a strong preference for arginine at the peptide C terminus. Analysis of natural peptides bound to RT1.A^a by both pool sequencing and anhydrotrypsin chromatography revealed that TAP polymorphism determined the presence or absence of arginine as the peptide C-terminal residue. This result highlights the in vivo impact of TAP–peptide selectivity, and provides evidence against a high rate of generation of new C termini by protease activity in the endoplasmic reticulum.