The strong association of the human MHC class I allele HLA-B27 with the development of the chronic inflammatory disease ankylosing spondylitis (AS) is clear and has been known for over three decades. Despite this, it is far from clear how HLA-B27 is directly involved in AS. In recent years considerable progress has been made in defining the assembly pathway and the protein components involved in successfully folding MHC class I molecules in the environment of the endoplasmic reticulum. This process involves a number of critical interactions, which may influence how HLA-B27 molecules fold and what peptides become loaded. The impact o the unpaired Cys-67 residue in the peptide-binding groove upon the behaviour of both correctl folded and misfolded HLA-B27 molecules, especially its ability to allow the formation of B27 heavy-chain oligomers or dimers, which may form novel targets for immune receptors, or be an indicator of intracellular stress, has also been the focus of much research. In this chapter we aim to review recent data to determine whether any biochemical features of HLA-B27 can supply clues as to its enigmatic role in AS and will also comment on future potential directions of biochemical research into HLA-B27.