In neuronal and hormonal release, regulated exocytosis requires an essential set of proteins: the soluble N-ethylmaleimide sensitive-factor attachment receptor proteins (SNAREs) syntaxin 1, SNAP-25, VAMP, and their regulator, Munc18. Recently, it was found that Munc18-1 can interact with syntaxin 1 through distinct mechanisms: an inhibitory mode enveloping syntaxin (mode 1), sequestering it from SNARE protein interactions, and direct binding to an evolutionarily conserved N-terminal peptide of syntaxin (mode 2/3). The latter interaction has been proposed to control "priming" of the fusion reaction, defined using electrophysiology, but it is unknown how this interaction is regulated, and any dynamic effect at the molecular or vesicular level in cells remains undiscovered. We now show that a phosphorylation site in syntaxin 1 (Ser14) regulates the N-terminal interaction with Munc18-1. Probing syntaxin 1 association with Munc18-1, in real-time and in living cells, we found that modification of Ser14 modulated the dynamics of this interaction, specifically at the plasma membrane. Destabilization of this dynamic interaction enhanced vesicle immobilization at the plasma membrane with a resulting inhibition of exocytosis. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.