Action potential duration at 50% of the amplitude (APD50) and peak force of isometric contractions were recorded from right ventricular trabeculae of rat heart at 26 degrees C. When the preparation was in the steady state at 0.2 Hz, a varied test interval was interposed. The premature contraction after a test interval of 0.25 s showed reduced force and increased APD50. Gradual prolongation of the interval from 0.25 to 80 s led to a gradual increase of force and shortening of APD50. At longer intervals force decreased and APD50 increased. This gave a negative APD50-force correlation. In the presence of the Ca2+ antagonists nifedipine or Mn2+, force was reduced to about 70%. APD50 was shortened to about 50% and was almost independent of the test interval. The slope of the negative APD50-force relationship was only 20% of that under control conditions. It is concluded that the inward Ca2+ current, isi, is a prerequisite for the interval-dependent changes in APD50 and for the negative APD50-force relationship. A reduction of the stimulation frequency from 0.2 to 0.003 Hz resulted in a gradual reduction of force and prolongation of APD50. Both force and APD50 were then almost independent of interposed test intervals. In low [Na+], at 0.2 Hz, force was almost maximal and APD50 was short at all test intervals. Thus, throughout these two interventions all changes in force were associated with opposite changes in APD50. The causal factor underlying the negative APD50-force relationship is probably an intracellular release of Ca2+ which activates the contractile filaments and inactivates isi.