We have been using ATP analogues to try and map two ATP binding sites in the α subunit of pig kidney Na,K-ATPase (Cavieres, 2000; Ward & Cavieres, 1998, 2003). In the native enzyme, TNP-8N₃-[α³²P]-ADP anchors at αLys480, near the FITC site; the FITC-enzyme incorporates the radioactive analogue at a downstream site instead. Here we focus on the conditions for photoinactivating and labelling both native and FITC-modified Na,K-ATPase with TNP-8N₃-ADP, specially the effects of Na⁺ and K⁺. The native sodium pump can be photoinactivated by TNP-8N₃-ADP in the presence of just 50 mM Tris/Cl^– (pH 7.5), 1 mM EDTA. The rate constant (k_inact) increases hyperbolically with k_inact(max) = 0.054 ± 0.002 min^-1 and K_{D(TNP-8N3-ADP)} = 0.11 ± 0.02 μM (mean ± S.E.); ATP and TNP-ATP protect with high affinities. Addition of Na⁺ does not affect k_inact but K⁺ decreases it, with K_0.5(K) = 12 ± 4 μM. Sodium ions reverse the K⁺ effect, and K_0.5(Na) is 0.58 ± 0.08 mM at 30 μM K⁺. All this is to be expected, as both native and FITC-modified Na,K-ATPase spontaneously adopt an E1 or ‘Na⁺ form’, whilst K⁺ causes a conformational shift towards a ‘K⁺-occluded form’, both cations acting at intracellular sites (Rephaeli et al., 1986). In the FITC-enzyme the surviving K⁺-phosphatase and P_i-phosphorylation reactions cannot be photoinactivated in the presence of Tris alone, not even at 200-fold higher TNP-8N₃-ADP concentrations. In this case, Na⁺ promotes photoinactivation hyperbolically with similar k_inact(max) but with K_0.5(Na) = 97 ± 9 mM; K⁺ counters this effect with K_0.5(K) = 2.5 ± 0.6 mM (Ward & Cavieres, 1998). As the intracellular Na⁺ and K⁺ affinities in the FITC-enzyme are similar to those in native Na,K-ATPase (Rephaeli et al., 1986), the latter Na⁺ and K⁺ effects can only be attributed to an external cation site. The hyperbolic Na⁺ activation may result from occupation of the ‘third external Na⁺ site’ (Cavieres & Ellory, 1975). It is then possible that Na,K-ATPase can shuttle K⁺ (and Na⁺?) plus unloaded cation sites in the absence of ATP and P_i; the forms that react with TNP-8N₃-ADP would be E1 (or E1null;Na_i⁺) and E2null;Na_o⁺ in the native and FITC-modified enzymes, respectively. To be commensurate with k_inact(max), the slippage needs to be about 0.02% of the active transport rate.