It has been shown that, in glycerinated skeletal muscle fibers, polyclonal antibody directed against myosin subfragment 2 (S-2) eliminates Ca(2+)-activated isometric force development, while Mg-ATPase activity of the fibers remains unchanged. To further explore possible involvement of myosin S-2 in muscle contraction, we studied the effect of anti-S-2 antibody on rigor linkage formation in the fibers. It was found that the antibody inhibited development of rigor force in a dose- and time-dependent manner, without changing the relation between muscle fiber stiffness and force. If, however, the antibody was applied after development of rigor force, it had no effect on both the stiffness and force. These results strongly suggest bidirectional functional communication between myosin subfragments 1 and 2 in the fibers.