An N-ethylmaleimide-sensitive fusion protein (NSF) has been purified on the basis of its ability to catalyse vesicular transport within the Golgi stack. We report here that this same protein is required for transport from the endoplasmic reticulum to the Golgi stack in semi-intact cells. This transport process is inhibited by a monoclonal antibody against NSF. Furthermore, pretreatment of semi-intact cells with N-ethylmaleimide, a sulphydryl alkylating reagent, inhibits transport. Addition of highly purified NSF largely restores transport from endoplasmic reticulum to Golgi. These results suggest that NSF is a general component of the transport machinery required for membrane fusion at multiple stages of the secretory pathway.