Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein and plays a role in reverse cholesterol transport by promoting cellular cholesterol efflux and activating lecithin-cholesterol acyltransferase. Notwithstanding these favourable properties, apoA-I has been found to be deposited as amyloid fibrils in vivo in the arterial intima and in atherosclerotic plaque tissues. Methionine residues are linked to the pathogenicity of several amyloid diseases as they are one of the most readily oxidized amino acids. Up to 25% of circulating serum apoA-I is found with at least one of its three methionine residues oxidised. Oxidation of the methionine residues of apoA-I results in destabilisation of the oligomeric structure of the protein, leading to formation of fibrillar aggregates that display all the hallmarks of amyloid. However, the mechanism by which methionine oxidation contributes to fibril formation is still not fully elucidated. Specifically, the site-specific contribution of each methionine residue towards the destabilisation and fibrillisation of apoA-I upon oxidation remains unknown. Mutant forms of apoA-I have been constructed substituting leucine for methionine at each of the three methionine positions. Using these constructs the specific contribution of oxidation of each specific methionine residue is assessed. Oxidation of methionine residue 148 appears to provide the largest contribution to the destabilisation of apoA-I, when compared to oxidation at methionine residues 86 and 112. This difference is proposed to stem from the respective location of each of the residues within the apoA-I helical bundle structure. This work highlights an important role for methionine oxidation in the formation of amyloid fibril structure and gives new insight into mechanisms underlying amyloid formation.