Studies of the passive mechanical properties of cardiac tissue have traditionally been conducted at sub-physiological temperatures and at various concentrations of extracellular calcium ([Ca²⁺]_o). More recently, use has been made of the negative inotropic agent 2,3-butanedione monoxime (BDM). However, there remains a lack of data regarding the influence that temperature, calcium and BDM have on the passive mechanical properties of cardiac tissue. We have used the dynamic stiffness technique, a sensitive measurement of cross-bridge activity, in which minute (~0.2% of muscle length), sinusoidal perturbations were applied at various frequencies (0.2-100 Hz) to quiescent, viable, right-ventricular (RV) rat trabeculae at two temperatures (20 °C and 26 °C) and at two concentrations of extracellular calcium (0.5 mM and 1.25 mM), in both the presence and absence of BDM (20 mM). The stiffness spectra (amplitude and phase) were found to be temperature-and [Ca²⁺]_o- sensitive in the absence, but insensitive in the presence, of BDM. From the index of cross-bridge cycling (the ratio of high-frequency to low-frequency stiffness amplitude), we infer that BDM inhibits a small degree of spontaneous sarcomere activity, thereby allowing the true passive properties of trabeculae to be determined. In the absence of BDM, the extent of spontaneous sarcomere activity decreases with increasing temperature. We caution that the measured mechanical properties of passive cardiac tissue are critically dependent on the experimental conditions under which they are measured. Experiments must be performed either at sufficiently high temperatures (>25°C) to ensure a low resting concentration of intracellular Ca²⁺ or in the presence of an inhibitor of cross-bridge cycling.