In the sliding actin in vitro motility assay, movement of fluorescently-labeled actin filaments over myosin is visualized using a microscope. In order to directly visualize myosin and, thus, correlate actin's movement with its position on myosin, native thick filaments must be used. As our first step to study Limulus native thick filaments, we isolated myosin from Limulus striated muscle and examined the sliding velocity of actin filaments over the purified myosin. Limulus muscle is regulated by both the thin (troponin and tropomyosin) and thick (myosin light chain phosphorylation) filament regulatory system which are calcium-dependent. Our results demonstrated that the "off' state of either system is dominant, and for maximal movement of actin filaments to occur both phosphorylated Limulus myosin and an activated troponin and tropomyosin system is required. Tropomyosin from different sources appeared to increase the sliding velocity 5-10 fold when bound to actin. Calcium, on the other hand, does not alter the velocity significantly in the absence of both troponin and tropomyosin.