ATP-dependent, inorganic phosphate-supported45Ca2+uptake by digitonin-lysed adult rat ventricular cardiomyocytes was used to evaluate the effects of simulated ischemia and reperfusion on the physically intact sarcoplasmic reticulum. Mitochondrial reactions were inhibited with rotenone and oligomycin.45Ca2+accumulation in the presence of the calcium efflux inhibitors, procaine (10 mM) and ruthenium red (30 μM), was used to characterize unidirectional uptake kinetics. A decrease in pH from 7.2 to 6.6 increased the [Ca2+] K0.5from 0.5 to 2.0 μM and reduced the apparent Vmaxby 28%. In the absence of procaine and ruthenium red, at a free [Mg2+] of 0.5 mM, maximum net uptake occurred at pCa 6.2 when pH was 7.2 and at pCa 6.0 when pH was 6.6. At lower pCa, net Ca2+accumulation declined. Increasing free [Mg2+] from 0.5 to 1 mM at pH 6.6 or to 2.5 mM at pH 7.2 increased net45Ca2+accumulation in the absence of procaine and ruthenium and shifted maximum uptake to pCa 5.6 and 6.0, respectively. Increases in cytosolic free [Mg2+] thought to occur during myocardial ischemia are therefore capable of inhibiting calcium efflux from the sarcoplasmic reticulum. Reducing [ATP] from 10 to 1 mM reduced maximum net45Ca2+uptake by 30% both in the presence and absence of efflux inhibitors. Preincubation of intact myocytes under conditions designed to simulate ischemia and reperfusion decreased45Ca2+uptake ≥.50%. The data indicate that myocardial ischemia and reperfusion can alter both Ca2+accumulation and calcium release by the sarcoplasmic reticulum.