The soluble N-ethyl maleimide-sensitive factor attachment protein receptor machinery is involved in membrane docking and fusion. In this machinery, the syntaxin family is a central coordinator and participates in multiple protein-protein interactions. In this study we have shown that a-fodrin, nonerythroid spectrin, is a new binding partner of the syntaxin family. a-Fodrin bound to syntaxin-1a, -3, and -4, all of which are localized on the plasma membrane. Syntaxin-3 interacted with a-fodrin in dose-dependent and saturable manners but not with a-spectrin, erythroid spectrin. Syntaxin-3 interacted with a-fodrin through its C-terminal coiled-coil region. Binding of Munc18 or SNAP-25 to syntaxin-1a inhibited the interaction of a-fodrin with syntaxin-1a. Available evidence indicates that a-fodrin is implicated in exocytosis, but a precise mode of action of a-fodrin in exocytosis remains unclear. Our results suggest that a-fodrin regulates exocytosis through the interaction with members of the syntaxin family.