Oligomeric α-synuclein (αS) is considered to be the potential toxic species responsible for the onset and progression of Parkinson's disease, possibly through the disruption of lipid membranes. Although there is evidence that oligomers contain considerable amounts of secondary structure, more detailed data on the structural characteristics and how these mediate oligomer–lipid binding are critically lacking. This report is, to our knowledge, the first study that aimed to address the structure of oligomeric αS on a more detailed level. We have used tryptophan (Trp) fluorescence spectroscopy to gain insight into the structural features of oligomeric αS and the structural basis for oligomer–lipid interactions. Several single Trp mutants of αS were used to gain site-specific information about the microenvironments of monomeric αS, oligomeric αS and lipid-bound oligomeric αS. Acrylamide quenching and spectral analyses indicate that the Trp residues are considerably more solvent protected in the oligomeric form compared with the monomeric protein. In the oligomers, the negatively charged C-terminus was the most solvent exposed part of the protein. Upon lipid binding, a blue shift in fluorescence was observed for αS mutants where the Trp is located within the N-terminal region. These results suggest that, as in the case of monomeric αS, the N-terminus is critical in determining oligomer–lipid binding.