The stiffness of the sarcomeres was studied during the diastolic interval of 18 stimulated (0.5 Hz) cardiac trabeculae of rat (pH 7.4; temperature = 25°C). Sarcomere length (SL) and force (F) were measured using, respectively, laser diffraction techniques (resolution: 4 nm) and a silicon strain gauge (resolution: 0.63 μN). Sinusoidal perturbations (frequency = 500 Hz) were imposed to the length of the preparation. The stiffness was evaluated from the corresponding F and SL sinusoids by analysis of both signals together either in the time domain or in the frequency domain. A short burst (duration = 30 ms) of sinusoidal perturbations was repeated at 5 predetermined times during diastole providing 5 measurements of stiffness during the time interval separating two twitches. These measurements revealed that stiffness increases by ~30% during diastole, while a simultaneous expansion of the sarcomeres (amplitude = 10-60 nm) was detected. Measurements of the fluorescence of fura-2 under the same conditions revealed a continuous exponential decline of [Ca2+](i) from 210 to 90 nM (constant of time ~300 ms) during diastole. In order to test the possibility that the increase of sarcomere stiffness and the decline of [Ca2+](i) were coupled during diastole of intact trabeculae, we studied the effect of different free Ca2+-concentrations ([Ca2+]) between 1 and 430 nM on sarcomere stiffness in rat cardiac trabeculae skinned by saponin (n = 17). Stiffness was studied using 500 Hz sinusoidal perturbations of muscle length (ML). We found that, below 70 nM, the stiffness was independent of [Ca2+]; between 70 and 200 nM, the stiffness declined with increase of [Ca2+]; above 200 nM, the stiffness increased steeply with [Ca2+]. The data fitted accurately to the sum of two sigmoids (Hill functions): (1) at [Ca2+] < 200 nM the stiffness decreased with [Ca2+] (EC50 = 160 ± 13 nM; n = -2.6 ± 0.7) and (2) at [Ca2+]> 200 nM, stiffness increased with [Ca2+] (EC50=3.4 ± 0.3 μM; n = 2.1 ± 0.2) due to attachment of cross- bridges. From these results, it was possible to reproduce accurately the time course of diastolic stiffness observed in intact trabeculae and to predict the effect on stiffness of a spontaneous elevation of the diastolic [Ca2+]. Identical stiffness measurements were performed in 4 skinned preparations exposed to a cloned fragment of titin (Ti I-II) which has been shown to exhibit a strong interaction with F-actin in vitro. It was anticipated that Ti I-II would compete with endogenous titin for the same binding site on actin in the I-band. Below 200 nM, Ti I-II (2 μM) eliminated the Ca2+- dependence of stiffness. These results are consistent with the hypothesis that the Ca2+-sensitivity of the sarcomeres at [Ca2+] < 200 nM, i.e. where the myocytes in intact muscle operate during diastole, involves an association between titin molecules and the thin filament.