Cytoplasmic filaments, separated from the axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, support the directed movement of organelles in the presence of ATP. All organelles, regardless of size, move continuously along isolated transport filaments at 2.2 +/- 0.2 micron/sec. In the intact axoplasm, however, movements of the larger organelles are slow and saltatory. These movements may reflect a resistance to movement imposed by the intact axoplasm. The uniform rate of all organelles along isolated transport filaments suggests that a single type of molecular motor powers fast axonal transport. Organelles can attach to and move along more than one filament at a time, suggesting that organelles have multiple binding sites for this motor.