Exocytosis involves the formation of a fusion pore that connects the lumen of secretory vesicles with the extracellular space. Exocytosis from neurons and neuroendocrine cells is tightly regulated by intracellular [Ca²⁺] and occurs rapidly, but the molecular events that mediate the opening and subsequent dilation of fusion pores remain to be determined. A putative Ca²⁺ sensor for release, synaptotagmin I (syt), binds directly to syntaxin and SNAP-25, which are components of a conserved membrane fusion complex. Here, we show that Ca²⁺-triggered syt•SNAP-25 interactions occur rapidly. The tandem C2 domains of syt cooperate to mediate binding to syntaxin/SNAP-25; lengthening the linker that connects C2A and C2B selectively disrupts this interaction. Expression of the linker mutants in PC12 cells results in graded reductions in the stability of fusion pores. Thus, the final step of Ca²⁺-triggered exocytosis is regulated, at least in part, by direct contacts between syt and SNAP-25/syntaxin.