The aim of the present work was to analyse the interaction between Na +,K +-ATPase and one of our recent synthesized coumestans, namely the original molecule 2-methoxy-3,8,9-trihydroxy coumestan (PCALC36). Rat brain (mainly α2 and α3 Na +,K +-ATPase isoforms) and kidney (α1 isoform) fractions enriched with Na +,K +-ATPase were utilized to compare the inhibition promoted by PCALC36 with that of classical inhibitors like ouabain and vanadate. Analysis of inhibition curves revealed that unlike ouabain, which was about a thousand times more potent to inhibit brain isoforms than kidney isoform, PCALC36 had a similar affinity for brain ( ic 50=4.33±0.90 μM) and kidney ( ic 50=11.04±0.86 μM) isoforms. The inhibitory effect of PCALC36 was not antagonized by 1–10 mM K +, as observed with ouabain. Whereas vanadate was more potent in ionic conditions promoting the E2 conformation of the enzyme, the inhibitory effect of PCALC36 was equal in ionic conditions favouring either the E1 or E2 conformations. Equilibrium binding assays with [ 3 H ]ouabain revealed that the addition of 2–10 μM PCALC36 did not change the K d of ouabain but decreased its maximal binding ( B max) in a concentration-dependent manner (from 76.6 to 44.0 pmol/mg protein). This inhibitory effect of PCALC36 was not reverted after an extensive washing procedure indicating that it forms a very stable complex with Na +,K +-ATPase. We conclude that PCALC36, a new molecule with a non-steroidal skeleton, inhibits the Na +,K +-ATPase by a mechanism of action different from the cardiac glycosides and could thus serve as a structural paradigm to develop new inotropic drugs.