A series of 17 gitoxigenin 16β-formates, acetates, and methoxycarbonates was synthesized, including their 3β-acetates, formates, and digitoxosides. A 16β-formate group was generally found to increase activity 30 times, a 16β-acetate group 9–12 times, while a 16β-methoxycarbonate decreased activity by two-thirds. 3β-Formates and acetates had little effect on activity by themselves, but sometimes reduced the activity-increasing properties of 160-formates and acetates. A 3β-digitoxoside increases the activity of gitoxigenin by 15 times, but the effect is less if the 160-group is esterified. And finally, a 16-one decreases activity dramatically. These data suggest an important role for C16 esters and possibly the presence of a separate binding site on Na+,K+-ATPase corresponding to the cardenolide C16 position.