Conformational heterogeneity of alpha-synuclein was studied with electrospray ionization mass spectrometry by analyzing protein ion charge state distributions, where the extent of multiple charging reflects compactness of the protein conformations in solution. Although alpha-synuclein lacks a single well-defined structure under physiological conditions, it was found to sample four distinct conformational states, ranging from a highly structured one to a random coil. The compact highly structured state of alpha-synuclein is present across the entire range of conditions tested (pH ranging from 2.5 to 10, alcohol content from 0% to 60%), but is particularly abundant in acidic solutions. The only other protein state populated in acidic solutions is a partially folded intermediate state lacking stable tertiary structure. Another, more compact intermediate state is induced by significant amounts of ethanol used as a co-solvent and appears to represent a partially folded conformation with high beta-sheet content. Protein dimerization is observed throughout the entire range of conditions tested, although only acidic solutions favor formation of highly structured dimers of alpha-synuclein. These dimers are likely to present the earliest stages in protein aggregation leading to globular oligomers and, subsequently, protofibrils.