Motor proteins move unidirectionally along cytoskeletal polymers by coupling translocation to cycles of ATP hydrolysis. The energy from ATP is required both to generate force and to dissociate the motor-filament complex in order to begin a new chemomechanical cycle. For myosin, force production is associated with phosphate release following ATP hydrolysis, whereas dissociation of actomyosin is tightly coupled to the binding of ATP. Dynein, a microtubule motor, uses a similar cycle, suggesting that all cytoskeletal motors might operate by a common mechanism. Here we investigate kinesin's chemomechanical cycle by assaying microtubule movement by single kinesin molecules when intermediate states in the hydrolysis cycle are prolonged with ATP analogues or inhibitors. In contrast to myosin and dynein, kinesin with bound ADP dissociates from microtubules during translocation, whereas kinesin with unhydrolysed nucleotide remains tightly associated with the polymer. These findings imply that kinesin converts ATP energy into mechanical work by a pathway distinct from that of myosin or dynein.