Abstract: SNARE-dependent membrane fusion requires the disassembly of cis-SNARE complexes (formed by SNAREs anchored to one membrane) followed by the assembly of trans-SNARE complexes (SNAREs anchored to two apposed membranes). Although SNARE complex disassembly and assembly might be thought to be opposing reactions, the proteins promoting disassembly (Sec17p/Sec18p) and assembly (the HOPS complex) work synergistically to support fusion. We now report that trans-SNARE complexes formed during vacuole fusion are largely associated with Sec17p. Using a reconstituted proteoliposome fusion system, we show that trans-SNARE complex, like cis-SNARE complex, is sensitive to Sec17p/Sec18p mediated disassembly. Strikingly, HOPS inhibits the disassembly of SNARE complexes in the trans-, but not in the cis-, configuration. This selective HOPS preservation of trans-SNARE complexes requires HOPS:SNARE recognition and is lost when the apposed bilayers are dissolved in Triton X-100; it is also observed during fusion of isolated vacuoles. HOPS thus directs the Sec17p/Sec18p chaperone system to maximize functional trans-SNARE complex for membrane fusion, a new role of tethering factors during membrane traffic.