(20R)-20, 22-Dihydrodigitoxigenin (3a) and (20S)-20, 22-dihydrodigitoxigenin (3b) were isolated from (20R, S)-20, 22-dihydrodigitoxigenin (3) by three fractional crystallizations each from ethyl acetate. The two diastereomers have distinct NMR spectra and similar (Na+,K+)ATPase inhibitory activities (I50= 1.1-1.4 X 10-5M)äabout 1/100 as active as digitoxigenin (1). Their activity compared with other cardenolide analogues suggests a passive geometric role for the 20(22) double bond in eliciting (Na+,K+)ATPase inhibition, keeping the lactone carbonyl in the proper orientation. (20S)-3β,14β-Dihydroxy-22-methylene-5β,14β-cardanolide (7a) was then synthesized from 3a, and(20R)-3β,14β-dihydroxy-22-methylene-5β,14β-cardanolide (7b) from 3b. They were found to be equivalently active in inhibiting (Na+,K+)ATPase, with I50values of 7.0 X 10-5M. Although it has been usually believed that the 14β-hydroxyl of cardenolides increases binding to the receptor, 2b (the 14-ene derivative of 7b) was more than twice as active (I50= 3.0 X 10-5) than either 7a or 7b.