The effect of perfusate [Ca2+] on the function of cardiac sarcoplasmic reticulum (CSR) was assessed by the oxalate-supported Ca2+uptake rate of ventricular homogenates of isolated rat hearts maintained in a modified Langendorif preparation. The total Ca2+pumping activity of the CSR was determined by using 20 FM ruthenium red or 625 μM ryanodine to close the CSR Ca2+release channel. The homogenate Ca2+uptake rate in the absence of ruthenium red or ryanodine decreased progressively with increasing perfusate [Ca2+] (25.7±1.2, 21.4±1.5, 17.2±1.1, and 163±1.2 [mean±SEMI nmol Ca2+· min−1· mg−1for hearts perfused for 5 minutes with 0.2, 1.4, 2.8, and 5.6 mM Ca2+, respectively;p=0.0001;n=8). This depression was not observed when Ca2+uptake was assayed in the presence of ryanodine or ruthenium red. Since the Ca2+uptake in the presence of ryanodine or ruthenium red is determined by the Ca2+-ATPase, this result suggests that perfusion with varying [Ca2+] did not affect the Ca2+-ATPase. The observed decrease in Ca2+uptake in the absence of ryanodine or ruthenium red is caused by an increased efflux through the ryanodine-sensitive Ca2+ release channel. When hearts perfused for 5 minutes with 0.2 or 5.6 mM Ca2+were reperfused for 10 minutes with 1.4 mM Ca2+, homogenate Ca2+uptake rates were restored to near control levels. These effects of perfusate Ca2+were not direct effects, because changes in the [Ca2+] of the homogenization medium did not alter the homogenate Ca2+uptake activity in either the presence or absence of ryanodine. The homogenate Ca2+uptake rates were unaffected by prior active loading of the CSR with Ca2+. These results suggest a regulatory role of perfusate Ca2+in increasing the open state of the ryanodine-sensitive Ca2+release channel that is distinct from the beat-to-beat regulation of Ca2+release from the CSR by Ca2+(Ca2+-induced Ca2+release).