Kinetic studies on a rat brain (Na+ + K+)-dependent ATPase (EC 3.6.1.3) preparation demonstrated high-affinity sites for ATP, with a Km near 1 mum, and low affinity sites for ATP, with a Km near 0.5 mM. In addition, the dissociation constant for ATP at the low affinity sites was approached through the ability of ATP to modify the rate of photo-oxidation of the enzyme in the presence of methylene blue; a value of 0.4 mM was obtained. The temperature dependence of the Km values in these two concentration ranges also differed markedly, and the estimated entropy of binding was +27 cal/degree per mol at the high affinity sites, whereas it was -20 cal/degree per mol at the low affinity sites. Moreover, the relative affinities of various congeners of ATP as of the K+ -dependent phosphatase reaction of the enzyme indicated an interaction at the low-affinity sites for ATP: ATP, ADP, CTP, and the [beta-gamma] -imido analog of ATP all competed with Ki values near those for the ATPase reaction at the low affinity sites. Conversely, the Km for nitrophenyl phosphate as a substrate for the phosphatase reaction was near its Ki as a competitor at the low-affinity sites of the ATPase reaction. These observations are incorporated into a reaction scheme with two classes of substrate sites on a dimeric enzyme, manifesting idverse enzymatic and transport characteristics.