SNARE proteins represent a family of related proteins that are thought to have a central role in vesicle targeting and fusion in all eukaryotic cells. The binding properties of the neuronal proteins synaptobrevin 1 (VAMP1), syntaxin 1, SNAP-25, and soluble N-ethylmaleimide-sensitive factor attachment protein (alpha-SNAP), have been extensively studied. We report here the first biochemical characterization of a nonneuronal SNARE complex using recombinant forms of the yeast exocytic SNARE proteins Snc1, Sso1, and Sec9 and the yeast alpha-SNAP homolog, Sec17. Despite the low level of sequence identity, the association properties of the yeast and neuronal complexes are remarkably similar. The most striking difference we have found between the yeast and neuronal proteins is that individually neither of the target membrane SNAREs (t-SNAREs), Sso1 nor Sec9, show any detectable binding to the synaptobrevin homolog, Snc1. However, as a hetero-oligomeric complex, Sec9 and Sso1 show strong binding to Snc1. The clear dependence on the Sso1-Sec9 complex for t-SNARE function suggests that regulating the formation of this complex may be a key step in determining the site of vesicle fusion. In addition, we have used this in vitro assay to examine the biochemical effects of several mutations in Sec9 that result in pronounced growth defects in vivo. As expected, a temperature-sensitive mutation in the region most highly conserved between Sec9 and SNAP-25 is severely diminished in its ability to bind Sso1 and Snc1 in vitro. In contrast, a temperature-sensitive mutation near the C terminus of Sec9 shows no defect in SNARE binding in vitro. Similarly, a deletion of the C-terminal 17 residues, which is lethal in vivo, also binds Sso1 and Snc1 normally in vitro. Interestingly, we find that these same two C-terminal mutants, but not mutants that show SNARE assembly defects in vitro, act as potent dominant negative alleles when expressed behind a strong regulated promoter. Taken together these results suggest that the C-terminal domain of Sec9 is specifically required for a novel interaction that is required at a step following SNARE assembly.