The ATPase [EC 3.6.1.3] activity solubilized from Bacillus megaterium membrane was purified by ammonium sulfate fractionation, protamine sulfate treatment, and DEAE-cellulose column chromatography. From the DEAE-cellulose column the activity was eluted in two fractions (Peaks I and II). Disc gel electrophoresis revealed that Peak I consisted of two components, a major component called ATPase I and a minor one called ATPase II, whereas Peak II contained only a single protein component corresponding to ATPase II. Both ATPases thus purified required either Ca++ or Mg++ for their activities. Most of the ATPase activity in the crude alkaline extract of the membrane could recombine with the alkali-treated membrane in the presence of Ca++ or Mg++. Upon recombination, part of the activity (10–40%) became protected from cold inactivation at 0°C. The purified ATPases could also recombine with the membrane in the presence of Ca++ though Mg++ was much less effective in this respect.