The effect of 15 min of global, normothermic ischemia on cardiac sarcoplasmic reticulum (SR) was investigated using the Ca2+ uptake rate and 3H-ryanodine binding of ventricular homogenates and isolated SR vesicles. Ischemia did not affect ryanodine binding in the homogenate, while it increased it in the isolated SR vesicles. Although ischemia decreased the homogenate oxalate-supported Ca2+ uptake rate, measured in the presence of high ryanodine to close the ryanodine-sensitive efflux pathway (+RY), its decrease of the Ca2+ uptake rate, measured in the absence of ryanodine (-RY), was more marked. This finding was also observed in the isolated SR. Although inhibition of the Ca-ATPase and its coupled Ca2+ uptake by thapsigargin proportionately decreased SR Ca2+ uptake -RY and +RY, ischemia decreased the Ca2+ uptake -RY proportionately more. This result suggested that there was a greater fraction of Ca2+ uptake activity in ryanodine-sensitive vesicles after ischemia. However, ischemia also reduced the yield of SR activity in the isolated SR fraction and the results could potentially be due to differential selection of ryanodine-sensitive and ryanodine-insensitive SR in the isolation procedure. We directly tested the hypothesis that ischemia changes the fraction of Ca2+ uptake activity in the ryanodine-sensitive vesicles by estimating the Ca-oxalate capacity measured +RY and -RY. Ischemia decreased the capacity -RY much more than +RY in the homogenate, indicating that more of the SR volume and Ca2+ uptake activity was in the ryanodine-sensitive vesicles after ischemia.