The accumulation of filamentous -synuclein (-S) is associated with Parkinson’s disease. It remains controversial as to the mode (antiparallel or parallel) of -S self-assembly and whether an exact alignment of the central hydrophobic region is essential. In the present study, we performed in vitro assembly using -S with or without the attachment of artificial leucine zippers (Zips) capable of forming either parallel or antiparallel coiled coils and included a spacer in one derivative. Results showed that Zips accelerate filament assembly in both the parallel and antiparallel fashions, that a precise alignment of the central hydrophobic region is not essential, and that the antiparallel pairs displayed the highest thioflavin T signals. More importantly, cells expressing Zip-fused -S, but not -S alone, formed -S immunopositive and thioflavin S-positive inclusions in 7 days. The results suggest that -S can assemble in both parallel and antiparallel modes but have a higher tendency to assemble in the latter mode and that cells overexpressing Zip-fused -S may be used to screen -S assembly inhibitors due to enhanced ability to form inclusions.—Jiang, P., Ko, L., Jansen, K. R., Golde, T. E., Yen, S.-H. Using leucine zipper to facilitate -synuclein assembly.