Covalent alterations of cardiac myofibrils by protein phosphorylation may alter the free Ca ++ -force relation in a way that would affect the relaxation properties of the heart independent of the sarcoplasmic free Ca ++ concentration. The clearest picture for such an alteration is emerging from studies of phosphorylation of troponin I. Troponin I is phosphorylated in situ as a specific response to β-adrenergic stimulation and in vitro phosphorylation of cardiac myofibrils results in a rightward shift of the free CA ++ -myofibrillar activation relation. The mechanism for this effect appears to be an alteration of calcium binding properties of myofibrillar TnC induced by phosphorylation of Tnl. This effect may be responsible, in part, for the relaxant effect of catecholamines. P-light chains of myosin are partially phosphorylated in beating hearts but there is as yet no clear indication that the covalent phosphate content of myosin changes with the physiological state of the heart. Vanadate, a transition state analogue of phosphate, present in most tissues including heart, may modulate relaxation of the heart by a non-covalent interaction with myosin. We found that vanadate ion inhibits isometric tension and immediate stiffness of chemically skinned heart muscle fibers with a Ki of 50 μM. Vanadate did not affect myofibrillar calcium binding or the Ca dependence of normalized isometric force development by the heart fibers. Vanadate also depressed shortening velocity of the chemically skinned fibers contracting with zero external load. Our data therefore indicate that vanadate may not only alter the affinity of cardiac myosin for actin but also the rate of cross-bridge turnover.