Ca shifts in the isolated, perfused ventricular muscle of guinea-pig hearts were investigated with the aid of 45Ca under the conditions of complete equilibration of preparations with the isotope-containing solution. In some series of experiments total Ca content was also measured by means of atomic absorption spectrophotometry. The content of 45Ca at the end of 70 min equilibration period during which the ventricles were stimulated at a rate of 60/min was 1.66 +/- 0.09 mmol/kg w.w. This content dropped to 0.42 +/- 0.09 mmol/kg w.w. within 4 min of rest. Contractile force also decreased to 21% of control. Both the content of 45Ca and contractile force returned to the pre-rest values within the 4 min of post-rest stimulation. The difference between the total Ca content in the rested and stimulated muscle was comparable to the respective difference in 45Ca contents. A significant linear correlation between this 45Ca fraction which was lost at rest and recovered during the post-rest stimulation, and contractile force was found under the following experimental conditions: I. post-rest recovery, II. decay during rest, III. post-rest stimulation at various rates. These results are consistent with the hypothesis proposing that this Ca fraction is involved in the frequency-dependent control of contractile force.