Rat heart sarcolemma was shown to hydrolyze ATP in the presence of Ca2+ or Mg2+; Ka values for Ca2+ and Mg2+ were in the range of 0.58-0.67 and 0.72-0.83 mM, whereas Vmax values were 33-38 and 21-28 mumol Pi/mg per hr, respectively. Both Ca2+ ATPase and Mg2+ ATPase showed low- and high-affinity sites for ATP; the Km value for the low-affinity sites for both enzyme activities was 300-325 microM, whereas Km values for high-affinity sites were 75-85 and 100-108 microM, respectively. The pattern of nucleotide hydrolysis in the presence of Ca2+ was found to be different from that with Mg2+. Although both high concentrations of ADP and Pi inhibited the enzyme activities, Mg2+ ATPase was more sensitive to ADP and less sensitive to Pi in comparison to Ca2+ ATPase. Storage of sarcolemma at about 0 degrees C showed a greater increase in ATP hydrolysis with Ca2+ than with Mg2+. The inhibitory effect of Mg2+ on Ca2+ ATPase, unlike that of Ni2+, Co2+, and Cu2+, was more than that on Mg2+ ATPase. Treatment of membranes with sodium dodecylsulfate or deoxycholate produced a greater reduction in Mg2+ ATPase than in Ca2+ ATPase. These results further support the view that Ca2+ ATPase and Mg2+ ATPase may be two separate enzymes in heart sarcolemma. It is suggested that Ca2+-dependent ATPase may be involved in opening calcium channels for the entry of calcium, whereas Mg2+ ATPase may serve as a Mg2+ pump mechanism for the efflux of magnesium from the cardiac cell.