SNARE proteins control intracellular membrane fusion through formation of membrane-bridging helix bundles of amphipathic SNARE motifs. Repetitive cycles of membrane fusion likely involve repetitive folding/unfolding of the SNARE motif helical structure. Despite these conformational demands, little is known about conformational regulation of SNAREs by other proteins. Here we demonstrate that hsc70 chaperones stimulate in vitro SNARE complex formation among the ER/Golgi SNAREs syntaxin 5, membrin, rbetl and sec22b, under conditions in which assembly is normally inhibited. Thus, molecular chaperones can render the SNARE motif more competent for assembly. Partially purified hsc70 fractions from brain cytosol had higher specific activities than fully purified hsc70, suggesting the involvement of unidentified cofactors. Using chemical crosslinking of cells followed by immunoprecipitation, we found that hsc70 was associated with ER/Golgi SNAREs in vivo. Consistent with a modulatory role for hsc70 in transport, we found that excess hsc70 specifically inhibited ER-to-Golgi transport in permeabilized cells.