Volatile anesthetics were demonstrated to decrease calcium sensitivity and maximal developed force of detergent-treated rat cardiac skinned fibers. To further investigate the possible mechanisms involved in the decrease of force production, stiffness measurements were performed at defined levels of activation with the use of quick length changes of 0.3 to 4% of initial muscle length in the absence and in the presence of 2 MAC of halothane, enflurane, or isoflurane. The results of various series of experiments suggest that these anesthetics have multiple sites of action on cardiac myofibrillar proteins: 1) they decreased active stiffness indicating a decreased number of attached force-generating cross-bridges; 2) they increased the stiffness/force ratio suggesting that the individual force developed by each cross-bridge was decreased during anesthetic exposure; and 3) they increased the time constant of force recovery, which is consistent with the decreased rate of ATP hydrolysis described by others. These changes in cross-bridges kinetics and efficiency may result from conformational changes in all the protein systems involved in force production, and especially actin-myosin attachment and detachment. However, the changes observed were small despite a relatively high concentration of anesthetics; therefore, they will probably participate only to a moderate extent in the overall negative inotropic effect of these agents.