We tested the hypothesis that slowing of shortening velocity generated by type IIB fibers from hindlimb-unweighted (HU) rats resulted from a reduced ATPase activity and/or a reduction in the relative content of myosin light chain 3f isoform content (MLC(3f)). After 2, 3, and 4 wk of HU, maximal unloaded shortening velocity (V(o)) of single permeabilized semimembranosus muscle fibers was determined by the slack test. Subsequently, the myosin heavy chain and the relative content of MLC were determined by SDS-PAGE. The ratio of MLC(3f) to MLC(2f) was determined by densitometric analysis. In addition, myofibrils were prepared from permeabilized fibers (soleus and semimembranosus muscles) and assayed for resting myosin ATPase and Ca(2+)-activated myosin ATPase. After HU, V(o) declined by 28-40% and the MLC(3f)/MLC(2f) ratio decreased by 32 to 48%. A significant correlation between the relative amount of MLC(3f) and V(o) was found (r = 0.48, P < 0.05). Resting myosin ATPase rates were not different between myofibrils prepared from corresponding muscles of control and HU rats (P = 0.86). Ca(2+)-activated myosin ATPase activities also were not different between myofibrils prepared from corresponding muscles of control and HU rats (P = 0.13). These data suggest that the slowing of maximal unloaded shortening velocity in type IIB fibers with HU is, at least in part, due to a relative change in the essential light chain composition, a decrease in the relative amount of MLC(3f) and most likely a concomitant increase in MLC(1f). However, this reduction in V(o) is independent of myosin ATPase activity.