alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1. A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1. The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1. However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1.