Syntaxins are thought to function during vesicular transport as receptors on the target membrane and to contribute to the specificity of membrane docking and fusion by interacting with vesicle-associated receptors. Here, syntaxin 5 (Syn5) was shown to be an integral component of endoplasmic reticulum-derived transport vesicles. This pool, but not the target, Golgi-associated Syn5 pool, was essential for the assembly of vesicular-tubular pre-Golgi intermediates and the delivery of cargo to the Golgi. The requirement for vesicle-associated Syn5 in transport suggests a reevaluation of the basis for operation of the early secretory pathway.