Calcium-independent regulation of the contractile proteins of cardiac muscle has been studied using hyperpermeable cells from rat ventricles and sections of quickly frozen rat hearts. These preparations have been used to study maximum calcium-activated force, myosin ATPase activity, and the maximum velocity of unloaded shortening. β-adrenergic activity increases the amount of force and the ATPase activity in accordance with the concentration of the V1 isozyme of myosin. V3 activity is decreased at the same time. In tissues containing only V1, there is no change in maximum velocity in response to β-adrenergic stimulation. These results indicate that β-adrenergic stimulation recruits V1 force generators and probably regulates a transition between a calcium unresponsive and a calcium responsive force generator.