Calcium ions play a crucial role in the activation of working ventricular myocardium, however the effect of calcium on relaxation has not been well established. Both the sarcoplasmic reticulum [SR] and the myofilaments play a major role in relaxation. We investigated the effects of isoproterenol, ryanodine, diseased states and two Ca²⁺-sensitizers: EMD 57033 and ORG 30029 on relaxation in isolated myocardium. Isoproterenol, which enhances SR function and desensitizes the myofilaments to Ca lf, shortened relaxation of both the twitch and the Ca²⁺ transient. Ryanodine and disease states such as cardiomyopathy and hypertrophy, which impair SR function, prolonged both the twitch and the Ca²⁺ transient. In this situation, relaxation became directly related to [Ca²⁺]_i cycling. Both EMD 57033 and ORG 30029 increased force of contraction, and diastolic force and prolonged twitch relaxation to a great degree with little effect on the Ca²⁺ transient.This would indicate that interactions at the level of the thin and thick filaments may affect relaxation without significant changes in intracellular calcium mobilization. We conclude that calcium mobilization can affect diastolic function in slowed muscle contractions, but time course changes of Ca²⁺ transients cannot be used alone to elucidate mechanisms of cardiac relaxation.