Kinesin is a two-headed, ATP-dependent motor protein that moves along microtubules indiscrete steps of 8 nm. In vitro, single molecules produceprocessive movement, motors typically take ~100steps before releasing from a microtubule . A central question relates tomechanochemical coupling in this enzyme: how many molecules ofATP are consumed per step? For the actomyosin system,experimental approaches to this issue have generated considerablecontroversy. Here we take advantage of theprocessivity of kinesin to determine the coupling ratio withoutrecourse to direct measurements of ATPase activity, which aresubject to large experimental uncertainties. Beads carrying singlemolecules of kinesin moving on microtubules were tracked with highspatial and temporal resolution by interferometry,. Statistical analysis of theintervals between steps at limiting ATP, and studies offluctuations in motor speed as a function of ATPconcentration, allow the coupling ratio to bedetermined. At near-zero load, kinesin moleculeshydrolyse a single ATP molecule per 8-nm advance. Thisfinding excludes various one-to-many andmany-to-one coupling schemes, analogous to thoseadvanced for myosin, and places severe constraints on models for movement.