1. 1.|The steady state levels of Na+-dependent phosphoenzyme (E-P) in the (Na+ + K+)-ATPase (EC 188.8.131.52) of rat brain, obtained from a time course study of phosphoenzyme formation at 4°C, were dependent on the concentration of Na+ in the reaction and were maximal in the presence of 64 mM Na+. The plot of phosphoenzyme vs. Na+ concentration gave a curve which on conversion to a double reciprocal plot (1/E-P vs. 1/Na+) gave a line with two breaks, yielding apparently three linear segments. This may be taken to indicate the presence of multiple Na+ sites for the formation of the phosphoenzyme. To test this hypothesis further, the following approach was taken. By making the assumption that the phosphoenzyme may represent bound Na+, it was possible to subject the data to rigorous multiple-site analysis by utilizing steady-state binding equations described by Klotz and Hunston (1971) (Biochemistry 10, 3065-3069), and by Scatchard (1949) (Ann. N.Y. Acad. Sci. 51, 660-672). The analysis of the data by these methods suggests that these may be three non-equivalent Na+ activation sites for the formation of Na+-dependent phosphoenzyme in the (Na+ + K+)-ATPase. The estimated intrinsic association constants (Ka) for activation by Na+ at each of the three sites were 3.4, 0.295, and 0.025 mM-1, respectively. 2. 2.|The steady-state level of Na+-dependent phosphoenzyme was reduced by 2 H2O (deuterated water) and Me2 SO (dimethylsulfoxide). This inhibition was reversed by increasing the concentration of Na+ in the reaction but remained constant over a time course at any given Na+ concentration. An analysis of the effect of 2 H2O on Na+-dependent phosphoenzyme formation in the presence of gradually increasing Na+ concentration also revealed the presence of three non-equivalent sites for Na+. The intrinsic association constants (Ka) for the activation of Na+ at each of the three sites in the presence of 2 H2 O were changed to 1.4, 0.232, and 0.033 mM-1, respectively, which suggests a differential effect of 2 H2 O on the three non-equivalent Na+ sites. 3. 3.|On statistical grounds (±2 S.E. of mean) a two non-equivalent site model also fits the data. In this case, the intrinsic association constants (Ka) were 2.44 and 0.041 mM-1 in H2 O medium, and 1.062 and 0.048 mM-1, in 2 H2 O medium, respectively, showing a differential effect of 2 H2 O on the two non-equivalent sites. 4. 4.|The inhibitory effect of 2 H2 O and Me2SO on the formation of Na+-dependent phosphoenzyme was maximal when the enzyme was allowed a contact with these agents prior to the addition of Na+ in the reaction. On the other hand, prior contact of the enzyme with Na+ reduced or abolished the inhibitory effect of 2 H2 O or Me2 SO. Prior contact of the enzyme with ATP also abolished the inhibition of Me2 SO. These results support the view that H2 O plays a regulatory role in the active center of the (Na+ + K+)-ATPase so that its presence tends to favor the E2 (K+-accepting) form of the enzyme whereas Na+ binds to at least two of its activation sites, in an apparently competitive manner with respect to H2 O, yielding the conformation suitable for the phosphorylation (E1). At the lowest affinity Na+ activation site for either the 2 or 3 non-equivalent site case the presence of H2 O may facilitate its binding. The results also suggest that both Na+ or ATP can independently shift the enzyme conformation to E1.