Heart sarcolemma has been shown to contain an ATPase hydrolyzing system which is activated by millimolar concentrations of divalent cations such as Ca2+ or Mg2+. Although Ca2+-dependent ATPase is released upon treating sarcolemma with trypsin, a considerable amount of the divalent cation dependent ATPase activity was retained in the membrane. This divalent cation dependent ATPase was solubilized by sonication of the trypsin-treated dog heart sarcolemma with 1% Triton X-100. The solubilized enzyme was subjected to column chromatography on a Sepharose-6B column, followed by ion-exchange chromatography on a DEAE cellulose column. The enzyme preparation was found to be rather labile and thus the purity of the sample could not be accurately assessed. The solubilized ATPase preparations did not show any cross-reactivity with dog heart myosin antiserum or with Na+ + K+ ATPase antiserum. The enzyme was found to be insensitive to inhibitors such as ouabain, verapamil, oligomycin and vanadate. The enzyme preparation did not exhibit any Ca2+-stimulated Mg2+ dependent ATPase activity. Furthermore, the low affinity of the enzyme for Ca2+ (Ka = 0.3 mM) rules out the possibility of its involvement in the Ca2+ pump mechanism located in the plasma membrane of the cardiac cell.