Sperm of the nematode, Ascaris suum, are amoeboid cells that do not require actin or myosin to crawl over solid substrata. In these cells, the role usually played by actin has been taken over by major sperm protein (MSP), which assembles into filaments that pack the sperm pseudopod. These MSP filaments are organized into multi-filament arrays called fiber complexes that flow centripetally from the leading edge of the pseudopod to the cell body in a pattern that is intimately associated with motility. We have characterized structurally a hierarchy of helical assemblies formed by MSP. The basic unit of the MSP cytoskeleton is a filament formed by two subfilaments coiled around one another along right-handed helical tracks. In vitro, higher-order assemblies (macrofibers) are formed by MSP filaments that coil around one another in a left-handed helical sense. The multi-filament assemblies formed by MSP in vitro are strikingly similar to the fiber complexes that characterize the sperm cytoskeleton. Thus, self-association is an intrinsic property of MSP filaments that distinguishes these fibers from actin filaments. The results obtained with MSP help clarify the roles of different aspects of the actin cytoskeleton in the generation of locomotion and, in particular, emphasize the contributions made by vectorial assembly and filament bundling.