Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. All vesicle transport steps also have an essential requirement for a member of the Sec1 protein family, including the neuronal Munc18-1 (also known as nSec1) in regulated exocytosis. Here, in adrenal chromaffin cells, we expressed a Munc18 mutant with reduced affinity for syntaxin, which specifically modified the kinetics of single-granule exocytotic release events, consistent with an acceleration of fusion pore expansion. Thus, Munc18 functions in a late stage in the fusion process, where its dissociation from syntaxin determines the kinetics of postfusion events.