Abstract The instability of the oxalate-supported Ca 2+ uptake activity of rat cardiac sarcoplasmic reticulum (CSR) in ventricular homogenates most likely accounts for the low specific activity of the rate of oxalate-supported Ca 2+ uptake in previously reported fractions of isolated rat CSR. We have found that CSR vesicles with improved Ca 2+ transport capabilities can be isolated if 1 m KCl is used to stabilize the CSR activity and to allow the extraction of the CSR from the cellular debris. The average rate of Ca 2+ uptake by the isolated rat CSR in the presence of 10 m m oxalate at 37°C was 0.45 μmol/min-mg in the absence of CSR Ca 2+ channel blockers and 0.87 μmol/min-mg in the presence of 10 μ m ruthenium red. The Ca 2+-dependent ATPase activity under the conditions of oxlate-supported uptake was 1.25 μmol/min-mg and 0.84 μmol/min-mg in the absence and presence of 10 μ m ruthenium red, respectively. The rat CSR vesicles bound 3H-ryanodine with a K d of 1.45 n m and a B max of 3.7 pmol/mg. The level of phosphorylated intermediate was 0.30 nmol/mg. The values of B max, EP and Ca 2+-ATPase activity are from one-third to one-half of those previously reported for isolated canine CSR vesicles. These results suggest that the isolated rat CSR may be quite similar to dog CSR.