Kinesin is a two-headed, ATP-dependent motor protein(1,2) that moves along microtubules in discrete steps(3) of 8 nm. In vitro, single molecules produce processive movement(4,5); motors typically take similar to 100 steps before releasing from a microtubule(5-7). A central question relates to mechanochemical coupling in this enzyme : how many molecules of ATP are consumed per step? For the actomyosin system, experimental approaches to this issue have generated considerable controversy(8,9). Here we take advantage of the processivity of kinesin to determine the coupling ratio without recourse to direct measurements of ATPase activity, which are subject to large experimental uncertainties(8,10-12). Beads carrying single molecules of kinesin moving on microtubules were tracked with high spatial and temporal resolution by interferometry(3,13). Statistical analysis of the intervals between steps at limiting ATP, and studies of fluctuations in motor speed as a function of ATP concentration(14,15), allow the coupling ratio to be determined. At near-zero load, kinesin molecules hydrolyse a single ATP molecule per 8-nm advance. This finding excludes various one-to-many and many-to-one coupling schemes, analogous to those advanced for myosin, and places severe constraints on models for movement.