OBJECTIVE The long-term purpose of this research was to establish the relationships between composition, structure, and function that affect human lens membranes. The authors hypothesized that the functional differences of epithelial, cortical, and nuclear lens membranes are related to compositional differences. Furthermore, age-dependent alterations in membrane function and structure can also be related to variations in the phospholipid composition. To explore these possibilities, the authors determined the phospholipid composition of epithelial, cortical, and nuclear membranes from pools of human lenses of different ages. METHODS Membranes were extracted from pools of clear human lenses of different ages using a monophasic methanolic extraction that minimizes the interfacial fluff produced with biphasic extractions. The phospholipid composition was determined by 31P NMR: RESULTS Only minor differences were detected between cortical and nuclear fractions. All phospholipids, except sphingomyelin, phosphatidylethanolamine, and the phospholipid with a shift of 0.12 parts per million (ppm) in the 31P NMR spectrum, showed significant differences in the epithelial fractions of all age groups compared to the fiber fractions; the percentage of phosphatidylcholine was considerably higher than that in the cortical and nuclear membranes of the same age. Conversely, the percentage of phosphatidylglycerol and lysophosphatidylglycerol was significantly smaller in the epithelial membranes than in the fiber membranes. The age-related changes in the composition of cortical and nuclear membranes were identical. These membranes showed a steady increase with age in the percentage of sphingomyelin and of an unidentified component with a shift of 1.2 ppm. The percentage of phosphatidylcholine decreased with age in both epithelial and fiber membranes. The rate of decrease was greater in the epithelial membranes than in the fiber membranes. Epithelial membranes contained approximately five times more phosphatidylcholine than fiber membranes of corresponding age. CONCLUSIONS Regardless of age, the composition of epithelial cell membranes was different than that of cortical and nuclear membranes, which showed similar phospholipid content. This suggests that significant compositional changes occur when epithelial cells become elongated to form fiber cells.