Synaptic transmission is a highly orchestrated process in which calcium triggers neurotransmitter release on a microsecond time-scale. Exocytosis requires the assembly of SNARE complexes between the vesicle SNARE synaptobrevin and the plasma membrane SNAREs, syntaxin and SNAP-25. Thus the regulation of SNARE complex assembly is a potentially important mechanism by which proteins can adjust synaptic strength. Tomosyn is a syntaxin-binding protein capable of regulating SNARE complex assembly in vitro through its SNARE binding domain. Tomosyn forms a stable complex with syntaxin and SNAP-25 that is virtually indistinguishable from SNARE complexes, based on X-ray crystallography. Tomosyn overexpression in endocrine cells leads to inhibition of dense core vesicle (DCV) fusion, suggesting tomosyn inhibits endocrine release. To test whether tomosyn regulates synaptic transmission we examined C. elegans tomosyn mutants. We have obtained two mutants in C. elegans tomosyn (tom-1). tom-1 mutants have mild behavioral defects indicating that TOM-1 is not an essential protein, however, pharmacological assays suggest that ACh transmission is enhanced. To elucidate the physiological basis of this phenotype we measured release at NMJs of dissected tom-1 mutants. Evoked responses in these mutants were significantly broader than wild-type, resulting in a two-fold increase in evoked charge integral. We attributed this effect to a change in presynaptic ACh release for the following reasons. 1) Neither the amplitude nor the kinetics of minis was altered, suggesting that post-synaptic reception is normal. 2) TOM-1 is enriched in presynaptic terminals based on immunohistochemistry. 3) We could reverse the tom-1 mutant synaptic phenotype, by expressing TOM-1 in cholinergic neurons. How might tomosyn regulate presynaptic release? Like its mammalian orthologs, the SNARE domain of TOM-1 complexes with syntaxin and SNAP-25, an interaction predicted to inhibit synaptic vesicle priming. We therefore, examined the responses of tom-1 mutants to hyperosmotic saline to measure the primed vesicle pool. The two-fold increase in hyperosmotic responses observed at tom-1 mutant synapses suggests that the number of primed vesicles was increased. We have recently shown that plasma membrane contacting vesicles represent a morphological correlate of priming, based on analysis of priming-defective unc-13 mutants. When we examined tom-1 mutant synaptic profiles, we observed a two-fold increase in morphologically contacting vesicles consistent with the enhanced release observed electrophysiologically. Conversely, overexpression of TOM-1 reduced both evoked responses and the number of morphologically docked vesicles. Based on these data we propose that tomosyn acts as a negative regulator of synaptic vesicle priming. Consistent with this conclusion, we found that tom-1 mutants partial suppressed unc-13 mutant priming defects, suggesting that TOM-1 antagonizes UNC-13-dependent priming, possibly by competing with synaptobrevin in SNARE complex assembly, thereby restricting vesicle fusion-competence. We next asked whether TOM-1 similarly regulates DCV release. DCV fusion requires CAPS, encoded by unc-31 in C. elegans. The paralysis of unc-31 mutants can be rescued by expressing UNC-31 in cholinergic neurons. To determine the physiological basis for the behavioral defects in unc-31 mutants we examined their NMJs. unc-31 mutants exhibited a 50% reduction in evoked release and a three-fold accumulation of DCVs, consistent with the proposed requirement of UNC-31 in DCV release. Conversely the number of DCVs in tom-1 mutants was reduced by 50%, where as TOM-1 overexpression caused DCV accumulations similar to unc-31 mutants. We attributed the loss of DCVs in tom-1 mutants to exuberant release, the replenishment of DCVs failing to keep pace. To examine whether there is a genetic interaction between unc-31 and tom-1 we generated double mutants. The behavioral defects of tom-1;unc-31 mutants were less pronounced than unc-31 alone. Consistent with this observation, NMJ evoked responses improved in the double mutants and the accumulation of DCVs observed in unc-31 mutants was partially suppressed. These data indicate that tom-1 negatively regulates CAPS-dependent DCV release. In summary we have demonstrated that both UNC-13-dependent synaptic vesicle priming and CAPS-dependent DCV release are negatively-regulated by tomosyn. On the basis of these results we propose that tomosyn, through its interactions with the SNARE protein syntaxin, restricts the priming processes of both secretory organelles.