Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V-MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor.A detailed understanding of chemo-mechanical transduction by the myosin family of molecular motors requires determining the structures of each of the intermediates in the myosin ATPase cycle and understanding how they are interconnected. Although a full understanding of how the myosin motor works requires knowledge of the crystallographic structures of both strong and weak actin-binding states, only the latter have been available until recently (1, 2). However, a structure of myosin V crystallized in the absence of nucleotide has now appeared, and it reveals atomic level details that are quite distinct from those of previous, weak binding structures (3). Most significant of these is a closure of the actin-binding cleft, caused by an approximation of the upper and lower 50-kDa domains. This would be predicted to enhance actin-binding affinity. Concomitantly nucleotide affinity was reduced because of a ϳ6.5-Å separation of the P loop from Switch I within the catalytic site. These structural features, in combination with kinetic studies on actin binding by nucleotidefree myosin V (4), have led to the suggestion that this new myosin structure represents a rigor-like conformation (3). If this is so, then it follows that between the weak binding structures previously crystallized and this new, rigor-like structure, there must also be a series of "intermediate," post-hydrolytic structures that contain within their catalytic sites ADP, magnesium, and/or phosphate. Our recent studies on the kinetics of nucleotide release from myosin V (5) are consistent with this conclusion in that they propose a series of distinct myosin-nucleotide conformations characterized by progressively higher actin affinity and progressively lower nucleotide affinity,where A is actin, M is myosin, and D is ADP. These states ...