(1) The fluorescence of eosin Y in the presence of (Na + + K +)-ATPase is enhanced by Mg 2+. The enhancement by Mg 2+ is larger than that obtained with Na + (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg 2+ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na +. (2) The Mg 2+-dependent enhancement of fluorescence can be reversed by K + as well as by ATP. In the presence of Mg 2+ + P i (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg 2+ and a low concentation of K + (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg 2+ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K +-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per 32P-labelling site in the presence of 5 mM Mg 2+ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg 2+ + 0.25 P i, the K d values are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg 2+, 150 mM Na +, the K d values are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg 2+, the addition of K + gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per 32P-labelling site). With 5 mM Mg 2+ + 150 mM K +, the affinities of the two binding sites become identical, at a K d of 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg 2+-form to the K +-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na + in the presence of Mg 2+ does not appreciably change the rate of conversion to the K +-form, giving a rate constant of about 110 s −. However, the addition of oligomycin in the presence of Mg 2+ + Na + had a profound effect: the rate of conversion to the K +-form was decreased by a factor of 2000 to about 0.063 s −1. This suggests that the conformation with Mg 2+ alone is different from the conformation with Na + alone. (6) The effects of K +, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per 32P-labelling site are bound to ATP sites.