We have developed a novel method for tracking microtubule rotation in three dimensions, which uses only one optical component, a prism, without modification of other aspects of a standard microscope. We applied our method to a conventional in vitro sliding assay by tracking streptavidin-coated quantum dots that are bound to a sparsely-biotinylated microtubule sliding across lawns of kinesin motors. Our method achieves nanometer accuracy and returns three-dimensional positional information. Using this method, we found that surface-attached Eg5 monomeric fragments (a member of the kinesin-5 sub-family of microtubule-based motors, which is essential for the assembly and maintenance of the bipolar spindle architecture in vivo) drove counterclockwise rotation of sliding microtubules around their axis. These corkscrewing motions have not been seen previously for kinesin-5, and it demonstrates that single kinesin-5 heads produce torsional force as well as axial sliding force. We also found that the rotational pitch was insensitive to microtubule geometry [1]. This short-pitch rotation by single-headed kinesin-5 molecules is strikingly similar to both that of a plus-end directed, non-processive single-headed kinesin-1 molecules [1, 2], which have a N-terminal motor domain, and that of a minus-end directed, non-processive double-headed kinesin-14 molecules [3], which have a C-terminal motor domain. A value of ∼ 0.3 μm for the rotational pitch generated by theses three motors appears to represent a characteristic signature for non-processive motors. This suggests the possibility that a kinesin head possesses in common an inherent torque component. [1] Yajima J., Mizutani K. & Nishizaka T. Nat. Struct. Mol. Biol. (2008), [2] Yajima J. & Cross RA. Nat. Chem. Biol. 1 (2005) 338-41., [3] Walker RA., Salmon ED. & Endow SA. Nature 347 (1990) 780-2.