Phosphorylation by P-i of the Na,K-ATPase from rabbit kidney in the absence of Na+ ions but in the presence of Mg2+ ions has been studied. In the absence of K+ ions, unphosphorylated and phosphorylated states induce different fluorescence levels in the membrane-bound styryl dye RH421, and hence transitions between the two states were monitored. Transient kinetic studies of phosphorylation were initiated by manual addition of P-i or by photochemical release of P-i from 1-(2-nitrophenyl)ethyl phosphate (caged P-i) using laser flash photolysis at 308 nm. Equilibrium studies of phosphorylation showed that the apparent K-m for P-i was 23.0 +/- 0.3 mu M (mean +/- sem) at pH 7.1 and 21 degrees C. The dye fluorescence increased in a biphasic manner on addition of 500 mu M P-i to the enzyme: a rapid phase (t(1/2) < 1 s) and a slower exponential phase at 0.059 +/- 0.003 s(-1). The rate of the rapid phase was studied by fast concentration-jump experiments and exhibited first-order kinetics in P-i up to 60 mu M. Fluorescence records vs time were exponential, and a plot of the rate constant versus [P-i] had a slope of 1.47 x 10(5) M(-1) s(-1) and ordinate ([P-i] = 0) intercept of 3.1 s(-1). Addition of 50 mM NaCl to the phosphorylated enzyme induced an exponential decay in the dye fluorescence from which a rate constant of 0.10 +/- 0.005 s(-1) was determined. These data were interpreted in terms of transformations between conformational states E(1) and E(2), and the phosphorylated state P-E(2) defined in the Post-Albers mechanism of the Na,K-ATPase [Lauger. P., (1991) Electrogenic Ion Pumps, Sinauer Associates Inc., Sunderland, MA] as follows: [GRAPHICS] The RH421 fluorescence of state P-E(2) was studied over the pH range 6-8.5. Fluorescence was greatest at pH 8.5 and lowest at pH 6.0 in a simple binding isotherm with pK 7.5. The apparent K-m for P-i rose cooperatively with increasing pH (pK(a) 8.6 and a Hill coefficient of 2), Therefore in the absence of monovalent metal ions, occupation of the cation (K+) binding sites by protons promotes phosphorylation by P-i.