Current single-molecule techniques do not permit the real-time observation of multiple proteins interacting closely with each other. We here report an approach enabling us to determine the single-molecule fluorescence resonance energy transfer (FRET) kinetics of multiple protein-protein interactions occurring far below the diffraction limit. We observe a strongly cooperative formation of multimeric soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, which suggests that formation of the first SNARE complex triggers a cascade of SNARE complex formation.