The measurement of fluorescent-labeled actin filament movement driven by mechanoenzymes (e.g., myosin) is an important methodology for the study of molecular motors. It is assumed that the filament velocity (Vf) is analogous to the unloaded shortening velocity (Vu) seen in muscle fibers. Methods are described to reproducibly quantitate the movement of these filaments and to select uniformly moving filaments and specify their Vf. Use of these techniques allowed comparison of Vf to literature values for Vu with regard to [ATP], [ADP], [Pi], pH, ionic strength (10-150 mM), and temperature (15-30 degrees C). Vf and Vu are quantitatively similar with respect to the effects of substrate and product concentrations and temperatures greater than 20 degrees C. However, Vf is more sensitive to decreases in pH and temperatures less than 20 degrees C than Vu. At ionic strengths of 50-150 mM, Vf and Vu exhibit similar ionic strength dependencies (decreasing with ionic strength). At ionic strengths less than 50 mM, Vf is markedly reduced. Results of experiments using adenosine 5'-O-(3-thiotriphosphate) suggest that increasing the number of weakly bound cross bridges does not seriously affect Vf. Thus, although Vf is a good analogue for Vu under certain conditions (elevated ionic strength and temperatures greater than 20 degrees C), under others it is not. The results of motility assays must be cautiously interpreted.