The activity of the ubiquitous Na+/K+-ATPase represents a substantial portion of the resting metabolic activity of cells, and the molecular activity of this enzyme from tissues of different vertebrates can vary several-fold. Microsomes were prepared from the kidney and brain of the rat (Rattus norvegicus) and the cane toad (Bufo marinus), and Na+/K+-ATPase molecular activity was determined. The membrane lipids surrounding this enzyme were isolated and phospholipids prepared. ‘Surface pressure/area’ isotherms were measured in monolayers for both membrane lipids and phospholipids using classic Langmuir trough techniques. Microsomal lipid composition was also measured. Whilst significant correlations were observed between membrane composition and Na+/K+-ATPase molecular activity, the strongest correlations were found between the molecular activity and parameters describing the packing of the surrounding membrane lipids and phospholipids. The influence of membrane lipid composition, especially membrane acyl composition, on the activity of a membrane protein mediated by physical properties of the lipids may represent a fundamental principle applicable to other membrane proteins.