Membrane proteins located on vesicles (v‐SNAREs) and on the target membrane (t‐SNAREs) mediate specific recognition and, possibly, fusion between a transport vesicle and its target membrane. The activity of SNARE molecules is regulated by several soluble cytosolic proteins. We have cloned a bovine brain cDNA encoding a conserved 117 amino acid polypeptide, denoted G olgi‐associated AT Pase E nhancer of 16 kDa (GATE‐16), that functions as a soluble transport factor. GATE‐16 interacts with N‐ethylmaleimidesensitive factor (NSF) and significantly stimulates its ATPase activity. It also interacts with the Golgi v‐SNARE GOS‐28 in an NSF‐dependent manner. We propose that GATE‐16 modulates intra‐Golgi transport through coupling between NSF activity and SNAREs activation.