Squid axoplasm has proved a rich source for the identification of motors involved in organelle transport. Recently, squid axoplasmic organelles have been shown to move on invisible tracks that are sensitive to cytochalasin, suggesting that these tracks are actin filaments. Here, an assay is described that permits observation of organelles moving on unipolar actin bundles. This assay is used to demonstrate that axoplasmic organelles move on actin filaments in the barbed-end direction, suggesting the presence of a myosin motor on axoplasmic organelles. Indeed, axoplasm contains actin-dependent ATPase activity, and a pan-myosin antibody recognized at least four bands in Western blots of axoplasm. An approximately 235-kDa band copurified in sucrose gradients with KI-extracted axoplasmic organelles, and the myosin antibody stained the organelle surfaces by immunogold electron microscopy. The myosin is present on the surface of at least some axoplasmic organelles and thus may be involved in their transport through the axoplasm, their movement through the cortical actin in the synapse, or some other aspect of axonal function.