The purpose of this study was to examine the movements of Ca2+into the myocardium from the extracellular space during the course of muscle contraction. Equilibration of the rabbit right ventricular wall with perfusate containing45Ca was measured by collecting effluent droplets over time. This protocol was carried out in a quiescent muscle and then repeated 15-20 minutes later in an identical fashion except that halfway through the collection period the muscle was stimulated to contract. We were thus able to quantitate the contraction-dependent changes in45Ca movements. In control experiments using [S8Co]EDTA as an extracellular space marker, we observed that contraction altered the volume of this space. This alteration in extracellular space was relatively small, and the quantitation of45Ca movements was corrected for this change. The addition of 1 μM Bay K 8644 to the perfusate stimulated tension and augmented Ca2+depletion from the extracellular space. The addition of 15 μM nifedipine to the perfusate significantly reduced both tension development and Ca2+depletion from the extracellular space of the muscle. Net contraction-dependent movement of Ca2+from the extracellular space into the myocardium under control conditions at 1 mM [Ca2+] was 10-14 μmol Ca2+/kg tissue wet wt/beat. This value indicates either a large contribution of Ca2+-induced Ca2+release from the sarcoplasmic reticulum and/or significant contribution of sarcolemmal bound Ca2+to excitation-contraction coupling in the rabbit ventricle.