Adenosine triphosphate (ATP) hydrolysis by unfractionated brain suspensions has been measured manometrically and chemically in bicarbonate buffer. Both magnesium and, less strongly, calcium accelerate ATP hydrolysis by brain suspensions. The effectiveness of low magnesium or calcium concentrations is decreased by relatively high ATP concentrations. The optimum pH for phosphate liberation is about 8. The ratio of carbon dioxide evolved to inorganic phosphate liberated depends on the pH. Evidence is given that the suspensions contain a Mg-activated ATPase which is strongly inhibited by calcium, a Ca-activated ATPase, and a Mg-activated PPase which is strongly inhibited by calcium but is different from the Mg-ATPase. These enzymes were not obtained in "latent" form nor activated by glutathione. Ethylenediamine tetraacetate can inhibit Ca-ATPase completely but is less effective on the Mg-ATPase and can release the latter from calcium inhibition. Other inhibitors have been tested.The ATPase activity of cerebral cortex of mouse, rat, cat, beef, and man has been measured by a standard procedure. The activity per unit weight decreases with increasing size of the animal parallel to the decrease in the oxygen uptake rate. Calculations show that the ATPase activity is high enough to release all high energy phosphate which could be produced by esterification coupled with respiration.