We have developed an electrophoretic cell suitable for single-molecule electrophoresis. The setup works for fluorescently labeled macromolecules by direct recording of their motion under an external electric field. The electrophoretic mobility of rodlike, polydisperse actin filaments (F-actin) were measured, as well as its dependence on the orientation of the filaments. A dipping effect is observed and quantitatively accounted for by the difference in hydrodynamic drag between motions along and perpendicular to the long axis of a filament. When averaged over all orientations, the mobility of F-actin in 50 mM KCl and 2 mM MgCl(2) is determined to be -(8.5+/-0.7) x 10(-5) cm(2)(V s). This method is also used to compare the mobility of F-actin and fd virus in a mixture of them. A reliable ratio of 1.26 is measured for fd virus to F-actin. The influence of the orientation dependent drag on electrophoretic mobility is discussed and a strategy for reliable measurement is proposed.