Myosin-Va is a molecular motor that may participate in synaptic vesicle cycling. Calpain cleaves myosin-Va in vitro at methionine 1141 in the tail domain. We show that intracellular proteolysis of myosin-Va occurs in rat cortical synaptosomes depolarized in the presence of calcium, evidenced by the formation of an 80 k polypeptide that co-migrates in SDS-PAGE with the 80 k fragment produced by the in vitro proteolysis of myosin-Va by calpain. Anti-myosin-Va antibody recognized this polypeptide in Western blots and immunoprecipitated it from synaptosome extracts. Calpastatin, a calpain-specific inhibitor, or leupeptin, a general cysteine protease inhibitor, suppressed or blocked formation of the 80 k polypeptide depending on membrane permeability. We conclude that myosin-Va undergoes intracellular proteolysis by endogenous calpain, when synaptosomes are depolarized in the presence of calcium, at the same cleavage site previously identified in vitro, thus, making it a target for calcium signaling during synaptic activation.