In the streaming cytoplasm of the Characean algae cell, the movement of organelles along actin bundles occurs at a striking rate of up to 60 microns s-1. To further characterize the molecular mechanisms responsible for this phenomenon, we have reconstituted the movement of actin filaments in vitro using defined biochemical components. We report that only a soluble cytoplasmic fraction devoid of organelles and filamentous material supports the movement of fluorescent-labeled actin filaments on glass at a rate of up to 60 microns s-1. This fraction also contains the K(+)-EDTA ATPase and the actin-activated Mg2+ ATPase activities characteristic of myosin proteins. Therefore, on the basis of these observations, we conclude that Nitella cells have a soluble pool of non-filamentous myosin molecules with the mechanochemical properties expected for a motor responsible for cytoplasmic streaming in vivo. The preparation and conditions described here should be useful for the purification of this translocator.