Munc18-1, a member of the Sec1/Munc18 (SM) protein family, is essential for synaptic vesicle exocytosis. Mund 8-1 binds tightly to the SNARE protein syntaxin 1, but the physiological significance and functional role of this interaction remain unclear. Here we show that syntaxin 1 levels are reduced by 70% in munc18-1 knockout mice. Pulse-chase analysis in transfected HEK293 cells revealed that Munc18-1 directly promotes the stability of syntaxin 1, consistent with a chaperone function. However, the residual syntaxin 1 in munc18-1 knockout mice is still correctly targeted to synapses and efficiently forms SDS-resistant SNARE complexes, demonstrating that Munc18-1 is not required for syntaxin 1 function as such. These data demonstrate that the Munc18-1 interaction with syntaxin 1 is physiologically important, but does not represent a classical chaperone-substrate relationship. Instead, the presence of SNARE complexes in the absence of membrane fusion in munc18-1 knockout mice indicates that Munc18-1 either controls the spatially correct assembly of core complexes for SNARE-dependent fusion, or acts as a direct component of the fusion machinery itself. © 2005 International Society for Neurochemistry.