Members of the syntaxin gene family are components of protein complexes which regulate vesicle docking and/or fusion during transport of cargo through the secretory pathway of eukaryotic cells. We have previously demonstrated that syntaxin 5 is specifically required for endoplasmic reticulum to Golgi transport (Dascher, C., Matteson, J., and Balch, W. E.(1994) J. Biol. Chem. 269, 29363-29366). To extend these observations we have now cloned a protein from rat liver membranes which forms a native complex with syntaxin 5. We demonstrate that this protein is the mammalian homologue to yeast Sly1p, previously identified as a protein which genetically and biochemically interacts with the small GTPase Ypt1p and Sed5p, proteins involved in docking/fusion in the early secretory pathway of yeast. Using transient expression we find that overexpression of rat liver Sly1 (rSly1) can neutralize the dominant negative effects of excess syntaxin 5 on endoplasmic reticulum to Golgi transport. These results suggest that rSly1 functions to positively regulate syntaxin 5 function.