Gomphoside, a 5 alpha-H cardiac glycoside isolated from Asclepias fructicosa, has an unique double glycosidic linkage to the aglycon through oxygen atoms at 2 alpha and 3 beta of the steroid. The 3'-axial hydroxyl of its conformationally rigid sugar residue appears to be the functional group responsible for its potent inotropic activity. With use of gomphoside as the model compound, the conformation of the flexible glycosidic linkage of the 5 beta-H cardenolides, digitoxigenin alpha-L-rhamnoside and digitoxigenin beta-D-digitoxoside, and the 5 alpha-H cardenolides, uzarigenin alpha-L-rhamnoside and uzarigenin beta-D-6-deoxyalloside, were investigated with the aid of computer graphics and conformational potential energy calculations. The relative inotropic potencies of these cardenolides can be accounted for by considering their active binding conformations with their potential energy distributions. The conformational distribution of the glycosidic moiety was postulated to be the major determinant of the biological activity of these cardenolides.