Syntaxin-1A is part of the SNARE complex that forms in membrane fusion in neuronal exocytosis of synaptic vesicles. Together with SNAP-25 the single-span transmembrane protein syntaxin-1A forms the receptor complex on the plasma membrane of neuroendocrine cells. Previous studies have shown that syntaxin-1A occurs in clusters that are different from lipid rafts in neuroendocrine plasma membranes. However, the interactions that promote these clusters have been largely unexplored. Here, we have reconstituted syntaxin-1A into lipid model membranes, and we show that syntaxin cluster formation depends on cholesterol in a lipid system that lacks sphingomyelin and therefore does not form liquid-ordered phases that are commonly believed to represent lipid rafts in cell membranes. Rather, the cholesterol-induced clustering of syntaxin is found to be reversed by as little as 1-5 mol % of the regulatory lipid phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2)), and PI-4,5-P(2) is shown to bind electrostatically to syntaxin, presumably mediated by the highly positively charged juxtamembrane domain of syntaxin. Possible implications of these results to the regulation of SNARE-mediated membrane fusion are discussed.