We describe the construction and operation of an optimized microscope for kinesin motility assays, using either microtubules (MTs) sliding over kinesincoated surfaces or the motility of kinesin-coated beads on immobilized single microtubules. A Zeiss Axiovert was modified by replacing the standard Zeiss condenser and mechanical stage with an improved arrangement that provides much better mechanical stability and very high resolution differential interference contrast (DIC) imaging of unstained microtubules using standard tungsten lamp illumination. The microscope can be used routinely for MT surface-sliding assays, using low-volume flow cells constructed by sandwiching together two cover slips. The design allows for the optional addition of optical trapping and high-resolution tracking hardware, for recording of singlemolecule mechanical transients.