Increases in stimulation frequency between 0.3 and 2 Hz decrease developed tension in rat, seemingly inconsistent with the explanation that an elevation of intracellular Na+-ion concentration is responsible for the positive force-frequency relationships observed in most species. Thus the force-frequency relationships were reevaluated in isolated atrial muscle of rat and mouse heart that show negative relationships, comparing them with ferret and guinea pig that show positive relationships. Ryanodine (2 nM, 45-min exposure) markedly reduced potentiated postrest contractions in all four species and caused a marked negative inotropic effect especially at low stimulation frequencies in rat and mouse, a moderate effect in ferret, but only a modest effect in guinea pig. In the presence of ryanodine, all four species showed positive force-frequency relationships. These results indicate that activator calcium in rat, mouse, and ferret cardiac muscle has a large ryanodine-sensitive component that shows a negative force-frequency relationship, masking a component for positive force-frequency relationships that exists in all species.