Tomosyn is a cytoplasmic protein that was shown to bind to Syntaxin1 and SNAP-25 through an R-SNARE domain, forming a complex that is almost identical in structure to the neuronal SNARE complex. Tomosyn inhibits exocytosis in various cell types and these effects were attributed to direct competition between tomosyn's SNARE domain and Synaptobrevin/VAMP. In the present study, we investigated the contribution of different domains of tomosyn to its activity. We show that a tomosyn mutant that lacks the entire SNARE domain is a potent inhibitor of vesicle priming, similar to the full-length tomosyn. The SNARE domain of tomosyn failed to inhibit exocytosis, indicating that this domain is not required for the inhibition. In contrast, over-expression of a N-terminally truncated mutant did not lead to inhibition of exocytosis although this mutant still bound to Syntaxin. Our results indicate that tomosyn can inhibit exocytosis independently of its SNARE interaction with Syntaxin and that the integrity of the WD40-domain is crucial for tomosyn's inhibitory function. Furthermore, we demonstrate that the entire N-terminal region of tomosyn, the WD40-repeats and the linker, is required for tomosyn's inhibitory effect.