VAMP/synaptobrevin is one of a number of v-SNAREs involved in vesicular fusion events in neurons. In a previous report, VAMP was shown to form a complex with synaptophysin and myosin V, a motor protein based on the F-actin, and that myosin V was then released from the complex in a Ca2+-dependent manner. Here, we found that VAMP alone is bound to myosin V in a Ca2+-independent manner, and determined that the globular tail domain of myosin V is its binding site. The syntaxin-VAMP-myosin V formed in the presence of Ca2+/calmodulin (CaM), In the absence of CaM, only syntaxin-VAMP, or VAMP-myosin V complex was formed. Our results suggest that VAMP acts as a myosin V receptor on the vesicles and regulates formation of the complex.