In a ‘physiologically sequenced’ contraction (PSC), which loads the isolated muscle preparation in a manner which approximates that of the intact heart, isometric relaxation precedes isotonic relaxation and occurs at minimum (‘end-systolic’) length. We studied the effects of initial muscle length, load, temperature, calcium, and isoproterenol on the isometric relaxation phase of physiologically sequenced contractions to define the determinants of the rate of isometric relaxation of rat left ventricular myocardium. At the baseline temperature (28 °C), relaxation was found to be nonexponential, and the maximum rate of decline of force (-dF/dtma,) was used to evaluate changes in relaxation. Three factors, shortening, end-systolic length, and total load, were examined as possible mechanical determinants of −dF/dtmax. We found that −dF/dtmax is linearly related to end-systolic muscle length for lengths below 94% of L--,x; −dF/dtmax is also strongly related to total load for lightly loaded contractions, but peaks at loads of approximately 80% of peak developed force and declines thereafter. Shortening is poorly correlated with −dF/dtmax. The slope of the linear portion of the relation between −dF/dtma′ and end-systolic length appears to be independent of muscle-loading conditions, and sensitive to factors known to alter relaxation.