Objective. The HLA-B27 allele is strongly associated with the group of inflammatory diseases known as the spondylarthritides (SpA). The aim of this study was to perform a large-scale, direct biochemical analysis of the HLA-B*2705 peptidome in order to identify candidates for mimicry between HLA-B27 peptides derived from cartilage proteins and arthritogenic bacterial sequences and to refine the consensus binding motif of this important allele. Methods. The peptides were recovered by recombinant expression of soluble HLA-B27 molecules secreted from cultured chondrocytic cells or HeLa cells. Analysis was based on capillary chromatography and tandem mass spectrometry in combination with stable isotope labeling with amino acids in cell culture or chemical labeling with iTRAQ to enhance the validity of the data. Results. Over 1,268 B27 peptides were identified, with 569 of them at high certainty, thus enabling better refinement of the B27 motif. This enabled the prediction of both short peptides and long peptides whose middle residues likely bulge out of the binding groove. Moreover, we identified a number of human B27 peptide sequences derived from human cartilage proteins, some of which are similar to common bacterial sequences. Conclusion. The peptides we identified may provide the missing link between bacterial infections and the resulting SpA. © 2010, American College of Rheumatology.