Myosin V is a cellular motor protein, which transports cargos along actin filaments. It moves processively by 36-nm steps that require at least one of the two heads to be tightly bound to actin throughout the catalytic cycle. To elucidate the kinetic mechanism of processivity, we measured the rate of product release from the double-headed myosin V-HMM using a new ATP analogue, 3-(7-diethylaminocoumarin-3-carbonylamino)-3-deoxy-ATP (deac-aminoATP), which undergoes a 20-fold increase in fluorescence emission intensity when bound to the active site of myosin V (Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., and White, H. D. Myosin V is an unconventional myosin that transports organelles such as vesicles in neurons and melanosomes in melanocytes along actin tracks (1). To do this, myosin V has evolved the ability to move processively on actin for several m, requiring many ATP hydrolysis cycles (2). The processivity is at least partially explained by a kinetic mechanism in which the slow rate of ADP dissociation is the rate-limiting step of ATP hydrolysis (3,4). This is in contrast to the myosin II mechanism, in which ADP dissociation is much faster and dissociation of actin from myosin occurs with each cycle of ATP hydrolysis. As a result, myosin V has a much higher duty cycle, and even single headed myosin V is in the strongly bound actomyosin-ADP state 80 -90% of the time during steady-state hydrolysis (3). It has also been suggested that additional mechanisms promoting processivity may involve gating of product release (5, 6). Thus, biochemical cycles of the lead and trail head keep out of synchrony, so one head of the molecule is always strongly attached (actomyosin or actomyosin-ADP). Biochemical (7), mechanical (5, 6, 8), and structural studies (9, 10) suggest that intramolecular strain accelerates ADP dissociation from the trail head and/or inhibits ADP dissociation from the lead head. The long lever arm of myosin V, which contains six calmodulin or light chain molecules, produces a 20 -25-nm power stroke that can be observed as two substeps of 15-20 and 5 nm (11,12).Biochemical evidence showing the interaction between the two heads is from kinetic data obtained by Rosenfeld and Sweeney (7), who observed biphasic dissociation of mdADP 5 from actomyosin V-HMM. In these single-mixing stoppedflow experiments, the rates of mdADP dissociation were measured by mixing myosin-mdADP complexes with actin in the presence of either a large excess of ATP or ADP that can act as a chase reagent, binding to nucleotide-free sites as they form. An alternative approach to measure the post-power stroke dissociation of ADP resulting from a single turnover of ATP hydrolysis is to use double-mixing stopped-flow measurements. In these experiments, the myosin is first mixed with ATP (or fluorescent analogues), incubated for a few seconds to allow the ATP to bind to the myosin and be hydrolyzed, and then mixed with actin. A power stroke is associated with P i dissociation from act...