The effect of 15 min of global, normothermic ischemia on 3H-ryanodine binding and the oxalate-supported Ca2+ uptake of cardiac sarcoplasmic reticulum (SR) was investigated in parallel using ventricular homogenates of isolated perfused rat hearts. Ischemia increased the Ca2+ efflux under the uptake assay conditions, as demonstrated by the greater stimulation of Ca2+ uptake by high concentrations of ryanodine (+RY) to close the SR Ca2+ channel. This effect was partially reversed by reperfusion. Ischemia depressed Ca2+ uptake rate -RY at free [Ca2+] of 0.4 microM and above, while the depression + RY was significant only above 10 microM Ca2+. We tested the hypothesis that inhibition of the Ca-ATPase alone, by adding thapsigargin or cyclopiazonic acid, could reproduce the effects of ischemia on the homogenate Ca2+ uptake rate. Thapsigargin or cyclopiazonic acid proportionally depressed Ca2+ uptake rate +RY and -RY and produced distinctly different effects of ischemia. Ischemia did not change the Bmax or Kd for equilibrium 3H-ryanodine binding, or the Hill coefficient or KCa for the [Ca2+]-dependence of equilibrium 3H-ryanodine binding. The rate of ryanodine binding, measured under the uptake conditions, was increased by ischemia and further increased by reperfusion. The effect of ischemia on the rate and extent of equilibrium binding to the high-affinity ryanodine binding site were unrelated to the highly reproducible effects on SR Ca2+ uptake rates measured in the homogenate.