A dual-wavelength fluorimeter was constructed, which used two light emitting diodes (LEDs) to excite the fluorescence dye RH 421 alternately with two different wavelengths. The ratio of the emissions at the two excitation wavelengths provided a drift-insensitive signal, which allowed detection of very small changes of the fluorescence intensity. Those small changes were induced by ion binding and release in conformation E 1 of the Na,K-ATPase. Titration experiments were performed to determine equilibrium dissociation constants (+ standard deviation) for each step in the complete binding and release sequence: 0.12 _+ 0.01 mM (Ez(K2) ~ KEI), 0.08 _+0.01 mM (KE 1 +--~ El), 3.0_+0.2 mM (NaE t ¢-~ Et), 5.2 _+ 0.4 mM (NazE 1 ~ NaE1) and 6.5 + 0.4 mM (Na3E 1 ~ NazEt) at pH 7.2 and T= 16 °C. These numbers show that the affinities of the binding sites exposed to the cytoplasm, are higher for K + than for Na + ions, similar to what was found on the extracellular side. The physiological requirement for extrusion of Na + from the cytoplasm, and for import of K + from the extracellular medium seems to be facilitated not by favorable binding affinities in state E t but by the two ATP-driven reaction steps of the cycle, Ez(K2) + ATP ---) KzE t . ATP and Na3E t -ATP ~ (Na3) Et-R which border the ion exchange reactions at the binding sites in conformation E l .