The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the Kmvalue for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Qc). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Qa) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.