Cordil-LND796 is a new cardiotonic glycoside under development. In rat brain microsomes where three isoforms of the Na,K-ATPase with differential affinities for cardiac glycosides have been identified, Cordil had higher affinity for the alpha 3 (IC50 = 0.02 microM) than for the alpha 2 (IC50 = 0.6 microM) and the alpha 1 (IC50 = 30 microM) isozymes. Cordil is potentially a selective inhibitor for both alpha 2 and alpha 3 Na,K-ATPase isoforms. Using inside out vesicles we have shown that Cordil binds to and inhibits Na,K-ATPase at an extracellular site. The dissociation kinetic rates (k-1) from the ATPase and the phosphatase activity (K-dependent dephosphorylation) of the Na,K-ATPase were similar for Cordil. Despite these similarities to ouabain comparison of the kinetics of the Na,K-ATPase inhibition by ouabain and Cordil revealed marked differences in their association rates (k+1 = 0.7 l mol-1 min-1 and k+1 = 6 x 10(-3) l mol-1 min-1 respectively) and their dissociation rates (k-1 = 1.3 +/- 0.2 x 10(-4) s-1 and k-1 = 69 +/- 7 x 10(-4) s-1 respectively). Both binding association and dissociation rates were enhanced for Cordil. These data are compatible with a stabilizing effect of Cordil on the E2P conformational state of Na,K-ATPase.