Translocation of intracellular organelles requires interaction with the cellular cytoskeleton, but the membrane and cytoskeletal proteins involved in movement are unknown. Here we show that highly purified synaptic vesicles from electric fish added to extruded squid axoplasm can show ATP-dependent movement. The movement is indistinguishable from that of endogenous vesicles and has a slight preference for the orthograde direction. In the presence of a nonhydrolyzable ATP analog, the synaptic vesicles bind to axoplasmic fibers but do not move. Elastase treatment of vesicles inhibits both binding and movement. We conclude that a protein component on the surface of cholinergic synaptic vesicles from electric fish is conserved during evolution and so can be recognized by the organelle-translocating machinery of the squid axon, resulting in ATP-dependent movement. Synaptic vesicles apparently retain the capacity for fast axonal transport, even after they reach their intracellular destination.