We have examined Na+,K+‐ATPase molecular activity and membrane fatty acid composition in the heart of six mammalian and eight avian species ranging in size from 30 g in mice to 280 kg in cattle and 13 g in zebra finches to 35 kg in emus, respectively. Na+,K+‐ATPase activity scaled negatively with body mass in both mammals and birds. In small mammals, the elevated enzyme activity was related to allometric changes in both the concentration and molecular activity (turnover rate) of Na+,K+‐ATPase enzymes, while in small birds, higher Na+,K+‐ATPase activity appeared to result primarily from an increased molecular activity of individual enzymes. The unsaturation index of cardiac phospholipids scaled negatively with body mass in both groups, while a significant allometric increase in monounsaturate content was observed in the larger mammals and birds. In particular, the relative content of the highly polyunsaturated docosahexaenoic acid (22:6n‐3) displayed the greatest variation, scaling negatively with body mass and varying greater than 40‐fold in both mammals and birds. Membrane fatty acid profile was correlated with Na+,K+‐ATPase molecular activity in both mammals and birds, suggesting a potential association between membrane lipid composition and the activity of membrane‐bound enzymes in the hearts of endotherms.