Studies of the passive mechanical properties of cardiac tissue have traditionally been conducted at subphysiological temperatures and various concentrations of extracellular Ca(2+) ([Ca(2+)](o)). More recently, the negative inotropic agent 2,3-butanedione monoxime (BDM) has been used. However, there remains a lack of data regarding the influence of temperature, Ca(2+), and BDM 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 (approximately 0.2% of muscle length) sinusoidal perturbations are applied at various frequencies (0.2-100 Hz) to quiescent, viable right ventricular rat trabeculae at two temperatures (20 degrees C and 26 degrees C) and at two [Ca(2+)](o) (0.5 and 1.25 mM) in the presence and absence of BDM (20 mM). The stiffness spectra (amplitude and phase) were sensitive to temperature and [Ca(2+)](o) in the absence of BDM but insensitive in the presence of BDM. From the index of cross-bridge cycling (the ratio of high- 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 at sufficiently high temperatures (>25 degrees C) to ensure a low resting concentration of intracellular Ca(2+) or in the presence of an inhibitor of cross-bridge cycling.