Syntaxins are a family of vesicular transport receptors that are involved in membrane traffic through both the constitutive and regulated secretory pathways. Syntaxins 1A/B,2,3, and 4 are principally associated with the plasma membrane. Two of the syntaxins, 1A and 1B, have been suggested to be the docking receptors for synaptic vesicles with the presynaptic membrane. The most distant member of the family, syntaxin 5, has been found in the Golgi region and has significant homology (35% identity) with Sed5p, an essential protein in yeast which is required for vesicular transport from the endoplasmic reticulum (ER) to the Golgi stack. Here we present evidence that syntaxin 5 performs an analogous function in ER to Golgi transport in mammalian cells. Transient expression of an hemagglutinin-tagged full-length clone of syntaxin 5 and a truncated mutant lacking the transmembrane domain inhibited the transport of vesicular stomatitis virus glycoprotein to the Golgi stack. Under these conditions, vesicular stomatitis virus glycoprotein accumulated in pre-Golgi intermediates, which were strongly enriched in syntaxin 5. Our results suggest that syntaxin 5 is the functional mammalian homologue of Sed5p and provides evidence for its role in regulating the potential targeting and/or fusion of carrier vesicles following export from the ER.