Associations between proteins present on neurotransmitter-containing vesicles and on the presynaptic membrane are thought to underlie docking and fusion of synaptic vesicles with the plasma membrane, which are obligate steps in regulated neurotransmission. SNAP-25 resides on the plasma membrane and interacts with syntaxin (a plasma membrane t-SNARE) and VAMP (a vesicle v-SNARE) to form a core protein complex thought to be an intermediate in a biochemical pathway that is essential for vesicular transport. We have now characterized a protein, Hrs-2, that interacts with SNAP-25. The binding of Hrs-2 to SNAP-25 is inhibited by calcium in the physiological concentration range that supports synaptic transmission. Furthermore, Hrs-2 binds and hydrolyses nucleoside triphosphates with kinetics that suggest that ATP is the physiological substrate for this enzyme. Hrs-2 is expressed throughout the brain and is present in nerve terminals. Moreover, recombinant Hrs-2 inhibits calcium-triggered 3H-noradrenaline release from permeabilized PC12 cells. Our results suggest a role for Hrs-2 in regulating secretory processes through calcium- and nucleotide-dependent modulation of vesicle-trafficking protein complexes.