Two calcium-stimulated ATPases at optimal pH values of 5.0 and 7.0, which are designated as acid and neutral Ca(2+) -ATPase, respectively, were found in the membrane fraction of human milk, and their enzymatic properties were studied. For maximal activity, neutral Ca(2+)-ATPase required 0.45 mM Ca ion, while acid Ca(2+) -ATPase required 207 mM Ca ion. Neutral Ca(2+) -ATPase activity was not enhanced by adding the Mg ion at more than 0.1 mM. Among the nucleotides, neutral Ca(2+) -ATPase showed a higher substrate specificity to GTP, CTP, ITP, and UTP than to ATP, while ATP was the best substrate for acid Ca(2+) -ATPase. Neutral and acid Ca(2+) -ATPases had apparent Km values of 0.361 and 0.192 mM, and Vmax of 186 and 178 μmol ATP hydrolyzed/mg of protein per min, respectively. Both Ca(2+) -ATPases were potently inhibited by fluoride, lanthanide, vanadate, and p-chloromercuribenzoate, and inactivated by EDTA, EGTA, and CDTA, but were unaffected by N-ethylmaleimide, NaN3, ouabain, oxidized glutathione, or oligomycin, and were inactivated by heating at 60°C for 10 min. These enzymes were concentrated in the membrane fraction of the cream and skim milk membrane.