The mechanism of the negative force staircase in thin rat ventricular trabeculae was investigated and compared with the positive force staircase in dog ventricular muscles of comparable diameter. Increasing stimulus frequency from 0.2 to 0.5 and 1 Hz resulted in a stepwise reduction of twitch amplitude that was demonstrated in both 1.25 and 2.5 mM external calcium concentration ([Ca]o). The negative staircase was associated with no change in the amplitude of postrest contraction or rapid-cooling contracture at either [Ca]o investigated. Hence, neither decreased loading nor overloading of the rat sarcoplasmic reticulum with calcium during the steady state can be invoked as a likely explanation for the negative staircase. The results are consistent with a frequency-dependent increase in the refractoriness of the sarcoplasmic reticular calcium release process or a decrease in the amount of trigger for calcium release, assuming that the amount of calcium present in the release pool is constant from one frequency to the next. In contrast to the rat, canine ventricular muscle exhibited a positive force staircase, the slope of which depended on [Ca]o, as well as a frequency-dependent increase in postrest contraction and rapid-cooling contracture. Data obtained from this series of experiments suggests that increased filling of the release pool within the sarcoplasmic reticulum with calcium underlies the inotropic effect of high-frequency stimulation in canine ventricular muscle.