Previous reports showed that cleavage of vesicle-associated membrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting -cells inhibited Ca²⁺-evoked insulin secretion. In these reports, the soluble N-ethylmaleimide-sensitive factor attachment protein target receptor proteins might have formed complexes, which preclude full accessibility of the putative sites for neurotoxin cleavage. In this work, VAMP-2 and SNAP-25 were effectively cleaved before they formed toxin-insensitive complexes by transient transfection of insulinoma HIT or INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A (BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca²⁺ (glucose or KCl)-evoked insulin release proportionate to the transfection efficiency (40-50%) and an accumulation of insulin granules. With the use of patch-clamp capacitance measurements, Ca²⁺-evoked exocytosis by membrane depolarization to 10 mV was abolished by TeTx (6% of control) but only moderately inhibited by BoNT/A (30% of control). Depolarization to 0 mV to maximize Ca²⁺ influx partially overcame BoNT/A (50% of control) but not TeTx inhibition. Of note, cAMP activation potentiated Ca²⁺-evoked secretion by 129% in control cells but only 55% in BoNT/A-transfected cells and had negligible effects in TeTx-transfected cells. These results indicate that, whereas VAMP-2 is absolutely necessary for insulin exocytosis, the effects of SNAP-25 depletion on exocytosis, perhaps on insulin granule pool priming or mobilization steps, could be partially reversed by higher levels of Ca²⁺ or cAMP potentiation.