The brain homogenate of guinea pig was fractionated and the nerve ending fraction (synaptosome) was separated. After the nerve ending fraction was treated with hypotonic medium, it was subfractionated into five components. The activities of Na+, K+-ATPase (EC 3.6.1.3], K+-phosphatase and Mg2+, Ca2+-ATPase in each fraction were estimated, and it was found that the subcellular distribution of these three enzymes was quite similar, the highest activities of all three being present in the same membraneous fraction which is considered to be mainly composed of the synaptic membrane. The optimal concentration of CaCl2 for ATP hydrolysis in the presence of 2mM MgCl2 and 10−4M EGTA was about 8×10−5M. The physiological significance of these three enzymes in the membranes is discussed.