We aim to reveal the molecular mechanism underlying the force response to stretch in the heart by examining the dependence of the rate of inorganic phosphate (Pi) release in response to stretch on the degree of calcium activation. Permeabilized trabeculae of rat heart are activated by photolytic release of ATP, at 20°C, in the presence of saturating (32µM), or half-saturating (1µM), calcium concentrations, at an initial sarcomere length of 1.9 or 2.1 µm. For trabeculae with an initial sarcomere length of 1.9 µm, the trabeculae are stretched to 2.1 µm during the isometric plateau, held at a constant length and returned to their original length. The rate of Pi release is determined by the fluorescence change associated with phosphate binding to a fluorescently-labelled phosphate binding protein diffused into the preparation. At an initial sarcomere length of 1.9µm, the isometric force at full activation was 50 kN.m^-2 compared to 25kN.m^-2 at half-maximal activation. During the isometric phase, Pi release is at a steady rate of 8.2 and 4.8s^-1 (assuming a myosin head concentration of 120µM) at 32 and 1µM calcium, respectively. During stretch, the rate of Pi release decreases markedly at both activation levels, to 1.0 and 1.5s^-1 respectively. Activation from an initial sarcomere length of 2.1µm produced an isometric force of 65 kN.m^-2 with a steady Pi release rate of 8.1s^-1 at maximal activation, and an isometric force of 48kN.m^-2 with a steady Pi release rate of 4.9s^-1 at half-maximal activation. The rates of Pi release during stretch and after the end of the stretch at low activation are equal to the rates of Pi release at high activation. This demonstrates that stretch induces activation of trabeculae from their half-activated state to a state that is fully activated. The stretch-induced activation is maintained during stretch and during the isometric period following stretch. Thus, Stretch contributes to activation of the trabecula's actomyosin ATPase in a calcium-independent manner.