The specific transfer of vesicles between organelles is critical in generating and maintaining the organization of membrane compartments within cells. Syntaxin 6 is a recently discovered member of the syntaxin family, whose constituents are required components of several vesicle trafficking pathways. To better understand the function of syntaxin 6, we generated a panel of monoclonal antibodies that specifically recognize different epitopes of the protein. Immunoelectron microscopy shows syntaxin 6 primarily on the trans-Golgi network (TGN), where is partially colocalizes with the TGN adapter protein AP-1 on clathrin-coated membranes. Additional label is present on small vesicles in the vicinity of endosome-like structures. Immunoprecipitation of syntaxin 6 revealed that it is present in a complex or complexes with alpha-soluble NSF attachment protein, vesicle-associated membrane protein 2, or cellubrevin and a mammalian homologue of VPS45, which is a member of the sec1 family implicated in Golgi to prevacuolar compartment trafficking in yeast. We show that mammalian VPS45 is found in multiple tissues, is partially membrane associated, and is enriched in the Golgi region. Converging lines of evidence suggest that syntaxin 6 mediates a TGN trafficking event, perhaps targeting to endosomes in mammalian cells.