The role of membrane lipid composition on the modulation of ouabain sensitivity of cardiac Na⁺K⁺-ATPase has been studied in vitro using several animal species. The animals can be grouped as ouabain-sensitive and ouabain-insensitive species. Ouabain-sensitive species (I₅₀; 0.5–2.2 μM) include sheep, marmoset, pig and the guinea pig, whilst rat and mouse form the ouabain-insensitive group (I₅₀; 100–105 μM). Although no species variation in the distribution of major phospholipid classes was observed, significant differences were apparent in the proportions of certain saturated and unsaturated phospholipid fatty acids. Thus, there was a marked increase in the relative proportion of docosahexaenoic (22:6, ω-3) acid in the Na⁺/K⁺-ATPase preparations from the rat and mouse compared to ouabain-sensitive species. Despite these differences, all animals had similar proportions of total saturated (∑SAT) and total unsaturated (∑Unsat) fatty acids. On the other hand, a good correlation between the unsaturation index of membrane lipids and I₅₀ value for ouabain was observed. It is proposed that acyl chain characteristics (unsaturation and/or chain length) rather than the head group of the phospholipid molecule play a major role in the modulation of Na⁺/K⁺-ATPase to inhibition by ouabain.