To investigate the possibility of cooperative interactions between the two myosin heads in muscle contraction, Ca2+-activated force development, K+-EDTA- and Mg2+-ATPase activities, muscle fiber stiffness, and the velocity of unloaded shortening were measured on partially p-PDM treated glycerinated muscle fibers, which contained a mixture of myosin molecules with zero, one and two of their heads inactivated. It was found that the magnitude of the Ca2+-activated isometric force development was proportional to the square of both K+-EDTA- and Mg2+-ATPase activities and also to the square of muscle fiber stiffness. If the two myosin heads in the glycerinated fibers are assumed to react independently with p-PDM, the above results strongly suggest that (i) each myosin molecule in the thick filaments can generate force only when its two heads do not react with p-PDM, (ii) muscle fiber stiffness is determined by the total number of native heads, and (iii) there is no cooperative interaction between the two myosin heads in catalyzing ATP hydrolysis.