Synapses vary widely in the probability of transmitter release. For instance, in response to an action potential the phasic synapses of the crayfish have a 100-1000-fold higher release probability than tonic synapses. The difference in release probability is attributed to differences in the exocytotic machinery such as the degree of “zippering” of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex. I used physiological and molecular approaches to determine if the zippered state of SNAREs associated with synaptic vesicles and the interaction between the SNARE complex and Complexin influence the probability of release at the synapse. I used three Botulinum neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by SNARE interaction (zippering) or open to proteolytic attack. Under low stimulation conditions, the light-chain fragment of botulinum B (BoNT/B-LC) but not BoNT/D-LC or tetanus neurotoxin (TeNT-LC) cleaved VAMP and inhibited evoked release at both phasic and tonic synapses. In addition, a peptide based on the C-terminal half of crayfish VAMP’s SNARE motif (Vc peptide) designed to interfere with SNARE complex zippering at the C-terminal end inhibited release at both synapses. The susceptibility of VAMP to only BoNT/B-LC and interference by the Vc peptide indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end with the C-terminal end exposed under resting conditions. I used a peptide containing part of the crayfish Complexin central α-helix domain to interfere with the interaction between Complexin and the SNARE complex. The peptide enhanced phasic evoked release and inhibited tonic evoked release under low stimulation but attenuated release at both synapses under intense stimulation. Therefore, Complexin appeared to exhibit a dual function under low synaptic activity but only promoted release under high synaptic activity. The results showed that the zippered state of the SNARE complex does not determine initial release probability as a similar zippered SNARE complex structure under resting conditions is common to both phasic and tonic synapses. However, Complexin may have a role in influencing the initial release probability of a synapse. Therefore, the interaction between the SNARE complex and Complexin is important for release but other factors contribute more significantly to synaptic strength.