The precise biochemical role of N-ethylmaleimide-sensitive factor (NSF) in membrane fusion mediated by SNARE proteins is unclear. To provide further insight into the function of NSF, we have introduced a mutation into mammalian NSF that, in Drosophila dNSF-1, leads to temperature-sensitive neuroparalysis. This mutation is like the comatose mutation and renders the mammalian NSF temperature sensitive for fusion of postmitotic Golgi vesicles and tubules into intact cisternae. Unexpectedly, at the temperature that is permissive for membrane fusion, this mutant NSF binds to, but cannot disassemble, SNARE complexes and exhibits almost no ATPase activity. A well-charaterized NSF mutant containing an inactivating point mutation in the catalytic site of its ATPase domain is equally active in the Golgi-reassembly assay. These data indicate that the need for NSF during postmitotic Golgi membrane fusion may be distinct from its ATPase-dependent ability to break up SNARE pairs.