Isolated cardiac muscles generate tension more quickly at higher levels of Ca(2+) activation. We investigated the molecular mechanisms underlying this effect in permeabilized rat myocardial preparations by measuring the rate of tension recovery following brief shortening/restretch perturbations. Separate series of experiments used Ca(2+)-activating solutions with different pH values (pH 6.75, 7.00, and 7.25) and different phosphate (P(i)) concentrations (0, 2.5, and 5.0 mM added P(i)) to modulate the recovery kinetics. Subsequent analysis showed that the rate of tension recovery correlated (P < 0.001) with the relative residual tension, that is, the minimum tension measured immediately after restretch normalized to the steady-state isometric tension for the experimental condition. This new finding suggests that the rate at which cardiac muscles develop force increases with the proportion of cross bridges bound to the thin filament and is strong evidence of cooperative contractile activation.