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Objective To characterize the alterations, as well as their mechanisms, induced in the HLA–B27–bound peptidome expressed in live cells by the natural ERAP1 polymorphisms predisposing to ankylosing spondylitis (AS): R528K and N575D/Q725R. Methods HLA–B*27:05–bound peptides were isolated from 3 human lymphoid cell lines expressing distinct ERAP1 variants differing at residues 528 and/or 575/725. The high-performance liquid chromatography–fractionated peptide pools were compared by mass spectrometry based on identity of molecular mass and chromatographic retention time. The relative amount of each shared peptide in any given cell line pair was estimated from the respective ion peak intensities. Peptide sequencing was also carried out by mass spectrometry. Results HLA–B27–bound ligands predominant in the context of the ERAP1 variant with K528 collectively showed higher molecular mass, higher frequency of N-terminal residues resistant to ERAP1, and bulkier residues downstream of the N-terminus, relative to peptides predominant in the R528 context. None of these differences were observed with ERAP1 variants differing at positions 575/725, but not at residue 528. Neither R528K nor N575D/Q725R altered the mean length of B*27:05-bound ligands. Conclusion The R528K, but not the N575D/Q725R, polymorphism alters the expression levels of many HLA–B*27:05–bound peptides, depending on the susceptibility of their N-terminal residues to trimming and depending on the size of the amino acid side chains at multiple positions downstream of the N-terminus. The significant alterations in the B*27:05 peptidome and the structural features of the peptides that determine their differential expression in distinct ERAP1 contexts account for the association of the R528K polymorphism with AS.