The central features of the mechanical cycle that drives the contraction of muscle are two translational steps: the working stroke, whereby an attached myosin crossbridge moves relative to the actin filament, and the repriming step, in which the crossbridge returns to its original orientation. Although the mechanism of the first of these is understood in some detail, that of the second has received less attention. Here, we show that repriming occurs after detachment of the crossbridge from the actin, rather than intervening between two actomyosin states with ATP bound [Eisenberg, E. & Greene, L. E. (1980) Annu. Rev. Physiol. 42, 293-309]. To discriminate between these two models we investigated the single-molecule mechanics of the myosin-actin interaction in the presence of ATP analogues such as GTP, for which the hydrolytic step itself limits the actomyosin GTPase rate to a much lower rate than for ATP. The lifetimes of bound states was proportional to 1͞ [GTP], indicating that during the bound period myosin was in the actomyosin rigor configuration. Moreover, despite the very low actomyosin GTPase, the rate of actin binding and formation of the rigor state was higher than with ATP; it follows that most interactions with actin result in the release of GTP and not of the products, GDP and phosphate. There was no significant movement of the actin during this interaction, so repriming must occur while myosin is dissociated, as in the original Lymn- Taylor T he mechanical events responsible for muscle contraction are coupled to the actomyosin ATPase cycle. According to the basic scheme of Lymn and Taylor (L-T) (1) (Fig. 1A), in the relaxed state (crossbridge 90°to the filament axes) the myosin (M) is bound to ADP (D) and phosphate (P). The binding of actin (A) to M⅐D⅐P 90 leads to the working stroke, accompanied by product release and the formation of the rigor AM 45 state. ATP (T) binds to AM to form AM⅐T, from which actin rapidly dissociates. Hydrolysis then takes place while myosin is dissociated from actin, and the mechanical repriming of the crossbridge, which positions it for the start of the next cycle, is associated with this step. Further research by Eisenberg and collaborators (2) added important details to the scheme. The concentration of Ca 2ϩ regulates the binding to actin of the so-called ''strong binding'' set of myosin states (M and M⅐D) but has little effect on the binding of the ''weak'' states (M⅐T and M⅐D⅐P). This finding suggested that these two sets of states bind to actin in different conformations (2), from which it was natural to infer that the transition between these conformations might correspond to the working stroke. Moreover, the free energy change between M⅐T and M⅐D⅐P was very small (3), and the two complexes bound to actin with similar affinities (4), at least 2 orders of magnitude weaker than that of the strong states. The actin association constant appeared to be a marker for the inferred two conformational states. In this case, as argued by Eisenberg and Greene (5), the logical...