The inhibitory effect of formylated cardiac steroids (gitaloxin and its derivatives) on guinea-pig heart Na, K-ATPase was compared to that of other cardiac steroids with various hydroxy substituents. The decreasing order of potency of aglycones at equilibrium was as follows: gitaloxigenin greater than digitoxigenin greater than ouabagenin greater than digoxigenin greater than gitoxigenin greater than diginatigenin. This sequence was different to the sequence of drugs hydrophobic character. The compounds with hydroxy groups in the vicinity of the lactone ring (gitoxigenin, diginatigenin) were less potent than the hydrophilic compound ouabagenin. We propose that intramolecular bounding between 16 beta-OH and the lactone ring contributes to the relatively low potency of gitoxigenin and diginatigenin. The formylation of 16 beta-OH increased the potency of gitoxigenin by a factor of 41. The formylated compound (gitaloxigenin) was 5-fold more potent than digitoxigenin. The 3 beta-glycosylation of digoxigenin lead to pseudo-irreversible inhibitors of Na, K-ATPase. The half-time to achieve the equilibrium (for 5 mumol/l) was equal to 54 s, 90 s and 108 s respectively for digoxigenin monodigitoxoside, digoxin and desacetyllanatoside C. However, at equilibrium the three glycosides were equipotent, suggesting the existence of steric effects at the sugar site of the receptor. The sequence of potency observed for monodigitoxosides, monodigitalosides and tridigitoxosides after 60 min incubation was similar to that observed for the corresponding aglycones. These results suggest that the strongly negative inductive group 16 beta-OCHO is tightly bound to Na, K-ATPase, possibly to the same receptor site than that which is thought forming hydrogen and ionic bonds with the lactone ring. They show that the high toxicity of gitaloxin in guinea-pig heart is likely due to its high potency as Na, K-ATPase inhibitor.