Reconstitution of cardiac and skeletal sarcoplasmic reticula (SR) Ca^(2+)-ATPases into liposomes requires different conditions for optimum enzymatic activity. In the presence of phosphatidylcholine (PC), maximum activity was obtained at a cholate/Ca^(2+)-ATPase/PC ratio (w/w) of 2/1/80 for cardiac SR Ca^(2+)-ATPase, and 4.5/1/40 for skeletal SR Ca^(2+)-ATPase. Reconstitution of cardiac Ca^(2+)-ATPase did not require the presence of oxalate in the reconstitution buffers, while this was essential for skeletal Ca^(2+)-ATPase. The maximum rates of Ca^(2+) uptake by reconstituted vesicles were 700 n㏖/㎎/min for cardiac Ca^(2+)-ATPase, and 960 n㏖/㎎/min for skeletal Ca^(2+)-ATPase. These findings suggest that Ca^(2+)-ATPases from cardiac and skeletal SR require different conditions for reconstitution, even though they have similar amino acid sequences (80% homology) and phospholipid compositions.