Intramembrane electric field strength is a very likely determinant of the activity of ion-transporting membrane proteins in living cells. In the absence of any transmembrane electrical potential or surface potential, its magnitude is determined predominantly by the dipole potential of the membrane's lipid components and their associated water of hydration. Here we have used a fluorometric method to quantify the dipole potential of vesicles formed from lipids extracted from kidney and brain of eleven different animal species from four different vertebrate classes. The dipole potential was compared with the fatty acid composition and with the Na⁺,K⁺-ATPase molecular activity of each preparation. The magnitude of the dipole potential was found to be relatively constant across all animal species, i.e. 236-334 mV for vesicles prepared from the total membrane lipids and 223-256 mV for phospholipids alone. The significantly lower value for phospholipids alone is potentially related to the removal of cholesterol and/or other common soluble lipid molecules from the membrane. Surprisingly, no significant dependence of the dipole potential on fatty acid composition was found. This may, however, be due to concomitant compensatory variations in lipid head group composition. The molecular activity of the Na⁺,K⁺-ATPase was found to increase with increasing dipole potential. The fact that the dipole potential is maintained at a relatively constant value over a wide range of animal species suggests that it may play a fundamental role in ensuring correct ion pump conformation and function within the membrane.