Despite controversy over their use and the potential for toxic side effects, cardiac glycosides have remained an important clinical component for the treatment for congestive heart failure (CHF) and supraventricular arrhythmias since the effects of Digitalis purpurea were first described in 1785. While there is a wealth of information available with regard to the effects of these drugs on their pharmacological receptor, the Na(+), K(+)-ATPase, the exact molecular mechanism of digitalis binding and inhibition of the enzyme has remained elusive. In particular, the absence of structural knowledge about Na(+), K(+)-ATPase has thwarted the development of improved therapeutic agents with larger therapeutic indices via rational drug design approaches. Here, we propose a binding mode for digoxin and several analogues to the Na(+), K(+)-ATPase. A 3D-structural model of the extracellular loop regions of the catalytic alpha1-subunit of the digitalis-sensitive sheep Na(+), K(+)-ATPase was constructed from the crystal structure of an E(1)Ca(2+) conformation of the SERCA1a and a consensus orientation for digitalis binding was inferred from the in silico docking of a series of steroid-based cardiotonic compounds. Analyses of species-specific enzyme affinities for ouabain were also used to validate the model and, for the first time, propose a detailed model of the digitalis binding site.