The release ofhormones and neurotransmitters, mediated byregulated exocytosis, can bemodifiedby regulationofthe fusion pore. The fusion poreisconsideredstable and narrowinitially, eventually leadingtothe complete mergerofthe vesicleandtheplasma membranes. Byusingthe high-resolutionpatch-clampcapacitancetechnique,westudiedsinglevesicles andasked whethertheSec1/Munc18 proteins, interacting with the membrane fusion-mediating SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, affect fusion pore properties. Munc18-1 mutants were transfected into lactotrophs to affect the interaction of Munc18-1 with syntaxin1 (Synt1) (R39C), Rab3A (E466K), and Mints (P242S). Compared withwild-type,Munc18-1 E466Kincreased the frequencyofthe fusion event. The latter two mutants increased the fusion pore dwell-time. All the mutants stabilized narrow fusion pores and increased the amplitude of fusion events, likely via preferential fusion of larger vesicles, since overexpression of Munc18-1 R39C did not affect the average size of vesicles, as determined by stimulated emission depletion (STED) microscopy. Single-molecule atomic force microscopy experiments revealed that wild-type Munc18-1, but not Munc18-1 R39C, abrogates the interaction between synaptobrevin2 (Syb2) and Synt1binarytrans-complexes. However, neither formofMunc18-1 affected the interactionofSyb2 withthe preformed binarycis-Synt1A-SNAP25B complexes. This indicates that Munc18-1 performs a proofing function by inhibiting tethering of Syb2-containing vesicles solely to Synt1 at the plasmalemma and favoring vesicular tethering to the preformed binary cis-complex of Synt1A-SNAP25B. The association of Munc18-1 with the ternary SNARE complex leads to tuning of fusion pores via multiple and converging mechanisms involving Munc18-1 interactions with Synt1A, Rab3A, and Mints. © 2011 the authors.