Tension generation can be studied by applying step perturbations to contracting muscle fibers and subdividing the mechanical response into exponential phases. The de novo tension-generating isomerization is associated with one of these phases. Earlier work has shown that a temperature jump perturbs the equilibrium constant directly to increase tension. Here, we show that a length jump functions quite differently. A step release (relative movement of thick and thin filaments) appears to release a steric constraint on an ensemble of noncompetent postphosphate release actomyosin cross-bridges, enabling them to generate tension, a concentration jump in effect. Structural studies [Taylor KA, et al. (1999) Tomographic 3D reconstruction of quick-frozen, Ca 2؉ -activated contracting insect flight muscle. Cell 99:421-431] that map to these kinetics indicate that both catalytic and lever arm domains of noncompetent myosin heads change angle on actin, whereas lever arm movement alone mediates the power stroke. Together, these kinetic and structural observations show a 13-nm overall interaction distance of myosin with actin, including a final 4-to 6-nm power stroke when the catalytic domain is fixed on actin. Raising fiber temperature with both perturbation techniques accelerates the forward, but slows the reverse rate constant of tension generation, kinetics akin to the unfolding/folding of small proteins. Decreasing strain, however, causes both forward and reverse rate constants to increase. Despite these changes in rate, the equilibrium constant is strain-insensitive. Activation enthalpy and entropy data show this invariance to be the result of enthalpyentropy compensation. Reaction amplitudes confirm a strain-invariant equilibrium constant and thus a strain-insensitive ratio of pretension-to tension-generating states as work is done.enthalpy-entropy compensation ͉ length step ͉ non-Arrhenius ͉ protein folding ͉ tension generation T he question of how the many asynchronously operating myosin motors generate tension and movement in muscle fibers is a critical issue in biology. Two different mechanisms have been proposed. In one, thermal fluctuations in position enable myosin heads (cross-bridges) to bind actin in a strained configuration to generate tension (1, 2). This rectification process can occur between detached and attached heads or by heads changing angle between subsites while attached to actin. For these thermal ratchet models to function at physiological rates, a sequence of multiple attached states is generally required, the stiffer the cross-bridge the larger the number of states. Heterogeneous mechanisms, some with sequential temperature-sensitive and -insensitive tension-generating transitions, have also been invoked (3-5). Power stroke mechanisms in which tension generation occurs as a single-step conformational change in attached cross-bridges offer an alternative mechanism e.g. (6-10). Other important aspects include the relationship between intermediate states of the ATPase cycle and tension generation....