Several enzyme complexes drive cellular movements by coupling free energy-liberating chemical reactions to the production of mechanical work^1–3. A key goal in the study of these systems is to characterize at the molecular level mechanical events associated with individual reaction steps in the catalytic cycles of single enzyme molecules. Ideally, one would like to measure movements driven by single (or a few) enzyme molecules with sufficient temporal resolution and spatial precision that these events can be directly observed. Kinesin, a force-generating ATPase involved in microtubule-based intracellular organelle transport^4–10, will drive the unidirectional movement of microscopic plastic beads along microtubules in vitro^4,9. Under certain conditions, a few (≤10) kinesin molecules may be sufficient to drive either bead movement or organelle transport. Here we describe a method for determining precise positional information from light-microscope images. The method is applied to measure kinesin-driven bead movements in vitro with a precision of 1–2 nm. Our measurements reveal basic mechanical features of kinesin-driven movements along the micro-tubule lattice, and place significant constraints on possible molecular mechanisms of movement.