We tested the hypothesis that lower specific force (force/cross-sectional area) generated by type II fibers from hindlimb-unweighted rats resulted from structural changes in myosin (i.e. a change in the ratio of myosin cross-bridges in the weak- and strong-binding state during contraction). In addition, we determined if those changes were age-dependent. Permeabilized semimembranosus muscle fibers from young adult and aged rats, some of which were hindlimb unweighted for 3 wk, were studied for Ca²⁺-activated force generation and maximal unloaded shortening velocity. Fibers were also spin labeled specifically at myosin Cys707 to assess the structural distribution of myosin during maximal isometric contraction using electron paramagnetic resonance (EPR) spectroscopy. Myosin heavy chain isoform (MHC) expression and the ratio of MHC-to-actin were evaluated in each fiber. Fibers from the unweighted rats generated 34% less specific force than fibers from weight-bearing rats (P<0.001), independent of age. EPR analyses showed that the fraction of myosin heads in the strong-binding structural state during contraction was 11% lower in fibers from the unweighted rats (P=0.019), independent of age. More fibers from unweighted rats co-expressed MHCIIB-IIX compared to fibers from weight-bearing rats (P=0.049). Unweighting induced a slowing of maximal unloaded shortening velocity and an increase in the ratio of MHC:actin in fibers from young rats only. These data indicate that altered myosin structural distribution during contraction and a preferential loss of actin contribute to unweighting-induced muscle weakness. Furthermore, the age of the rat has an influence on some parameters of changes in muscle contractility that are induced by unweighting.