Platelets function by secreting components necessary for primacy clot formation, This report describes an in vitro assay that measures alpha-granule secretion. Using permeabilized platelets, it is possible to recreate Ca2+-stimulated release of platelet factor 4 (PF4) that is ATP- and temperature-dependent. Though other divalent cat ions can replace Ca2+ (i.e., Sr2+, Mn2+, Zn2+), there is no effect of Ba2+. Analysis by electron microscopy indicates that the in vitro assay also mimics the cytoskeletal. rearrangements and granule centralization that occurs upon platelet activation in vivo. Antibody inhibition studies show that PF4 release requires the general membrane fusion protein N-ethylmaleimide-sensitive factor (NSF) and well as the target membrane SNAP receptors (t-SNAREs), syntaxin 2, 4, and SNAP-23, As shown by electron microscopy, the anti-t-SNARE antibodies block granule to target membrane fusion. This finding is unique in that it is the first report of a role for two syntaxins in the same exocytosis event.