The isolated C2 domains of otoferlin stimulate membrane fusion. Titrations of each isolated C2 domain, from 0 to 30 µM, demonstrate the ability of the otoferlin C2 domains C2B, C2C, C2D, C2E, and C2F to stimulate fusion between v- and t-SNARE proteoliposomes. The isolated C2 domains failed to stimulate membrane fusion between protein-free (PF) liposomes. E indicates that the experiment was performed under EGTA conditions.