It is well known that the rate of intracellular calcium ([Ca2+]i) decline is an important factor governing relaxation in unloaded myocardium. However, it remains unclear to what extent, under near physiological conditions, the intracellular calcium transient amplitude and kinetics contribute to the length-dependent increase in force and increase in duration of relaxation. We hypothesize that myofilament properties rather than calcium transient decline primarily determines the duration of relaxation in adult mammalian myocardium. To test this hypothesis, we simultaneously measured force of contraction and calibrated [Ca2+]i transients in isolated, thin rabbit trabeculae at various lengths at 37 degrees C. Time from peak tension to 50% relaxation (RT50(tension)) increases significantly with length (from 49.8+/-3.4 to 83.8+/-7.4 ms at an [Ca2+]o of 2.5 mM), whereas time from peak calcium to 50% decline (RT50(calcium)) was not prolonged (from 124.8+/-5.3 to 107.7+/-11.4 ms at an [Ca2+]o of 2.5 mM). Analysis of variance revealed that RT50(tension) is significantly correlated with length (P<0.0001). At optimal length, varying the extracellular calcium concentration increased both developed force and calcium transient amplitude, but RT50(tension) remained unchanged (P=0.90), whereas intracellular calcium decline actually accelerated (P<0.05). Thus, an increase in muscle length will result in an increase in both force and duration of relaxation, whereas the latter is not primarily governed by the rate of [Ca2+]i decline.