To investigate the role of the myosin hinge region in muscle contraction, we examined the contraction characteristics and Mg-ATPase activity of glycerinated muscle fibers prepared from rabbit psoas in the presence and absence of polyclonal antibody directed against the subfragment 2 (S-2) region of myosin. The antibody-induced reduction of Ca(2+)-activated isometric force was always accompanied by a parallel decrease of muscle fiber stiffness, so that the stiffness versus force relation remained unchanged by the antibody treatment. Force-velocity relations of the fibers, obtained by applying ramp decreases in force at steady isometric forces, indicated that the antibody had no effect on maximum shortening velocity or on the shape of force-velocity curves. Simultaneous measurements of Mg-ATPase activity and Ca(2+)-activated force showed that Mg-ATPase activity of the fibers remained unchanged despite the antibody-induced reduction of isometric force even to zero. These results indicate that when anti-S-2 antibody attaches to the S-2 region of myosin molecules, their heads still hydrolyze ATP but no longer contribute to both force generation and muscle fiber stiffness.