In studies of ischemia and reperfusion, a major experimental problem has been the inability to measure intracellular ionized calcium ([Ca2+])) in the intact heart. We have developed a new approach in which the bioluminescent calcium indicator aequorin is used to measure [Ca2+]i, in the isolated, coronary-perfused ferret heart. Aequorin is loaded into subepicardial myocytes of the left ventricle, and the signals are recorded simultaneously along with isovolumic left ventricular (LV) pressure at a constant pacing rate. This system shows 1) no attenuation or change of time course of LV pressure development or coronary perfusion pressure after aequorin loading; 2) consistent responses to physiological interventions and drugs; 3) individual aequorin and pressure signals that do not require signal averaging for analysis; and 4) [Ca2+]i, levels comparable with those reported in tissue or isolated myocyte cell preparations. During 5 minutes of hypoxia, diastolic [Ca2+)iand LV diastolic pressure increased while the systolic values of both [Ca2+]iand pressure decreased. The peak-to-peak systolic [Ca2+]iversus LV isovolumic pressure relation remained close to the control curve. In contrast, during 3 minutes of global ischemia, LV systolic and diastolic pressures fell rapidly, while [Ca2+]iincreased substantially. The [Ca2+]iversus pressure relations for both systole and diastole shifted to the right, indicating desensitization of the contractile apparatus to [Ca2+]i. These results provide evidence that different primary mechanisms determine the systolic and diastolic responses to acute hypoxia versus ischemia. During hypoxia, changes in [Ca2+]ihandling probably play a major role, while during ischemia, changes in the Ca2+sensitivity of the myofilaments appear to be of primary importance in the modulation of contractile dysfunction.