Highly purified vesicles of cardiac sarcolemma were prepared from a homogenate of canine ventricular muscle by density gradient centrifugation. The preparation showed an extremely high content of (Na+,K+)-ATPase. The steady state levels of Na+-dependent phosphoenzyme formation in the presence of Triton X-100 and the specific ouabain binding in the absence of Triton X-100 were, respectively, 773 and 907 pmol.mg-1 under the optimum conditions. On the other hand, the amount of Ca2+-dependent phosphoenzyme formed in the absence of Triton X--100 was less than 2 pmol.mg-1. This demonstrates that the preparation was virtually free of contaminant sarcoplasmic reticulum fragments. The preparation showed ATP-dependent Ca2+ uptake. Almost all the Ca2+ accumulated on the addition of ATP was rapidly released by the subsequent addition of NaCl. This finding gives evidence that the ATP-driven Ca2+ pump exists in the cardiac sarcolemma. The Ca2+ uptake was unaffected by 2 microM digitoxin, 1 microM monesin, and 200 microM dinitrophenol. These results exclude the possibility that transmembrane gradients of Na+ and H+ were involved in this Ca2+ uptake. The Ca2+ pump was activated by calmodulin. The concentration of calmodulin giving a half-maximum activation was 0.05 micrograms.ml-1, which is equivalent to 3 nM. This activation was removed by addition of trifluoperazine, a specific inhibitor of calmodulin.