Cochlear inner hair cells (IHCs) use Ca²⁺-dependent exocytosis of glutamate to signal sound information. Otoferlin (Otof), a C₂ domain protein essential for IHC exocytosis and hearing, may serve as a Ca²⁺ sensor in vesicle fusion in IHCs that seem to lack the classical neuronal Ca²⁺ sensors synaptotagmin 1 (Syt1) and Syt2. Support for the Ca²⁺ sensor of fusion hypothesis for otoferlin function comes from biochemical experiments, but additional roles in late exocytosis upstream of fusion have been indicated by physiological studies. Here, we tested the functional equivalence of otoferlin and Syt1 in three neurosecretory model systems: auditory IHCs, adrenal chromaffin cells, and hippocampal neurons. Long-term and short-term ectopic expression of Syt1 in IHCs of Otof ^−/− mice by viral gene transfer in the embryonic inner ear and organotypic culture failed to rescue their Ca²⁺ influx-triggered exocytosis. Conversely, virally mediated overexpression of otoferlin did not restore phasic exocytosis in Syt1-deficient chromaffin cells or neurons but enhanced asynchronous release in the latter. We further tested exocytosis in Otof ^−/− hippocampal neurons and in Syt1^−/− IHCs but found no deficits in vesicle fusion. Expression analysis of different synaptotagmin isoforms indicated that Syt1 and Syt2 are absent from mature IHCs. Our data argue against a simple functional equivalence of the two C₂ domain proteins in exocytosis of IHC ribbon synapses, chromaffin cells, and hippocampal synapses.