The biochemical link providing effective coordination between the mitochondrial ATP synthetic machinery and the contractile apparatus following transitions in cardiac work remains enigmatic. Studies were designed to determine whether activation of the actomyosin adenosinetriphosphatase (ATPase) is a necessary part of the signaling mechanism to the mitochondrial ATP synthase or whether a rise in cytosolic free Ca2+ is sufficient to activate the synthase. With the use of Langendorff-perfused rat hearts, cardiac work was varied via changes in perfusion pressure and by the inclusion of a beta-adrenergic agent. Furthermore, 2,3-butanedione monoxime and verapamil were used to vary independently either the activity of the actomyosin ATPase or the level of cytosolic free Ca2+. Determinations of the in vivo mitochondrial membrane potential [delta psi m; see Wan et al. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H445-H452, 1993] and its vectorial displacement during work transitions provide valuable information concerning direct activation of the ATP synthase and proton movement through the membrane domain of the synthase. Increased cardiac work in the presence of the beta-adrenergic agent resulted in a decrease in delta psi m. Addition of 2,3-butanedione monoxime decreased cardiac work but did not change delta psi m. The inclusion of verapamil resulted in similar decreases in cardiac work. However, delta psi m reversed back to a value observed under control, low-work conditions. These results in conjunction with data regarding levels of high-energy phosphates, free Mg2+, and adenosine 3',5'-cyclic monophosphate suggest a Ca(2+)-mediated increase in the activity of the ATP synthase.