Kinesin-1 is a dimeric motor protein that moves cargo processively along microtubules. Kinesin motility has been proposed to be driven by the coordinated forward extension of the neck linker (a approximately 12-residue peptide) in one motor domain and the rearward positioning of the neck linker in the partner motor domain. To test this model, we have introduced fluorescent dyes selectively into one subunit of the kinesin dimer and performed 'half-molecule' fluorescence resonance energy transfer to measure conformational changes of the neck linker. We show that when kinesin binds with both heads to the microtubule, the neck linkers in the rear and forward heads extend forward and backward, respectively. During ATP-driven motility, the neck linkers switch between these conformational states. These results support the notion that neck linker movements accompany the 'hand-over-hand' motion of the two motor domains.