Alpha-synuclein exists as at least two structural isoforms: a helix-rich, membrane-bound form and a disordered, cytosolic form. Here, we investigated the role of membrane-bound alpha-synuclein in the aggregation process. In a cell-free system consisting of isolated brain fractions, spontaneous and progressive aggregation of alpha-synuclein was observed in membranes starting at day 1, whereas no aggregation was observed in the cytosolic fraction in a 3-day period. The addition of antioxidants reduced the aggregation in the membrane fraction, implicating the role of oxidative modifications. When excess cytosolic alpha-synuclein was added to brain membranes, the rate of aggregation was increased, while the lag time was unaffected. Incorporation of cytosolic alpha-synuclein into membrane-associated aggregates was demonstrated by fractionation and co-immunoprecipitation experiments. In our recent study, we showed that mitochondrial inhibitors such as rotenone, induced alpha-synuclein aggregation in cells. In the present study using rotenone-treated cells, the earliest appearance of alpha-synuclein oligomeric species was observed in membranous compartments. Furthermore, alpha-synuclein-positive inclusions were co-stained with DiI, a membrane-partitioning fluorescent dye, confirming the presence of lipid components in alpha-synuclein aggregates. These results suggest that membrane-bound alpha-synuclein can generate nuclei that seed the aggregation of the more abundant cytosolic form.