The microsome fraction from pig brain was treated with NaI and extracted with Lubrol. The extract was treated batchwise with aminoethyl-cellulose or phospho-cellulose. Then, the extracts were charged to an AE-cellulose column, equilibrated with 14 mM veronal-HCI buffer, pH 7.1 and eluted with NaCl plus ATP at pH 6.6. Three peaks of activity were eluted and named α, β, and γ. The specific activities of the three peaks were very high (up to 7000 μmoles P1 liberation/mg protein/hr), and the activities were inhibited completely by ouabain. The labilities of the three peaks differed. On sodium dodecylsulfate polyacrylamide electrophoresis they gave a main band with an apparent molecular weight of 100,000. The molecular weights of native active ATPase [EC 3.6.1.3] in the α and γ peaks were both determined as 480,000 using gel chromatography on Sephadex G-200 and Sepharose 6B. The specific gravities of the α and γ peak fractions were 1.085 and 1.115, respectively, determined by the method of Mizuno et al. Their protein-lipid ratios were roughly 1: 6 and 1: 2 respectively, although the peak partly overlapped that of free Lubrol. These findings suggest that this enzyme contains only one subunit, with a molecular weight of 100,000, in an active molecule with a molecular weight of 500,000. The amount of phosphorylated protein formed from 32P-ATP (using a preparation with a specific activity of 5,600) was 7.8 μmoles/g protein, which corresponded to a molecular weight of 125,000 per mole of phosphate. This finding also supports the view that the enzyme contained only one subunit, although the existence of small subunits with, for example, a molecular weight of 10, 000, was not disproved completely.