Intracellular [Ca2+] ([Ca2+]i), intracellular Na+ activity (aiNa), and contraction have been monitored in single myocytes isolated from the ventricles of rat hearts. Some of these cells showed an increase in the size of the twitch as stimulation frequency was increased (positive force-frequency relationship), while others showed a decrease in the strength of contraction as the frequency of stimulation was increased (negative force-frequency relationship). In cells that showed a positive force-frequency relationship, increasing stimulation frequency resulted in increases in aiNa, diastolic [Ca2+]i, systolic [Ca2+]i, and the amount of Ca2+ that could be released from the sarcoplasmic reticulum by caffeine. The rate of decline of the [Ca2+]i transient and the twitch also increased as stimulation frequency was increased. In cells that showed a negative force-frequency relationship, increasing stimulation frequency had less effect on aiNa and had either no effect or decreased systolic [Ca2+]i with no change in the amount of Ca2+ that could be released from the sarcoplasmic reticulum using caffeine. The rate of relaxation of the [Ca2+]i transient and the twitch again increased as stimulation frequency increased. The pattern and time course of mechanical restitution was the same in both cell types. Although these data are essentially descriptive, it is consistent with the hypothesis that the final contractile response observed during changes of stimulation frequency may be dependent on how the Ca2+ loading of the preparation varies with stimulation frequency.