Forty kinds of bufotoxins and related compounds were tested for inhibition of Na⁺, K⁺-adenosine triphosphatase from guinea pig heart, and the structure-activity relationship has been discussed. The inhibitory activities of bufotoxins were dependent upon the dicarboxylic acid and amino acid components. The compounds having both the arginine and suberic acid moieties showed the higher inhibitory activities. The sulfates and glucuronides of cardiac steroids exhibited much less potency than the parent genins. The mode of inhibition was determined by means of the Dixon and Lineweaver-Burk plots.