2’(3’)-0-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5’-triphosphate (FEDA-ATP), a spectroscopic tool used for studying skeletal muscle myosin ATPase subfragment 1, was applied to Na+/K+- ATPase (EC 3.6.1.37). In contrast to the myosin subfragment, we found that FEDA-ATP is not a substrate for Na + /K + -ATPase. On the other hand, FEDA-ATP showed an affinity for both the low (E2, K<j = 2001MM) and the high (Ei, Kd = 22,«M) affinity ATP-binding sites. When the microscopic affinities of FEDA-ATP were used for calculating the macroscopic affinity in the overall reaction according to Kj = (KdEl*KdE2)1/2, the experimentally measured inhibition constant of 66,wM was obtained. To evoke irreversible binding inhibitors, FEDA-ATP was transferred in its chromium (III) and cobalt(III) complex analogs, which are suitable tools for labelling the ATP binding sites of Na + /K+ -ATPase in a specific way.