The organization and physical properties of lens lipid membranes made from the total lipids extracted from the human lens cortex and nucleus of 41-to 60-year-old donors were investigated using electron paramagnetic resonance (EPR) spin-labeling. Profiles of the phospholipid alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity were assessed across membranes and coexisting membrane domains. Lens lipid membranes prepared from the lens cortex and nucleus were found to contain two distinct lipid environments, which were assigned as the bulk phospholipid-cholesterol domain and the cholesterol bilayer domain (CBD). The alkyl chains of phospholipids were strongly ordered at all depths, indicating that the amplitude of the wobbling motion of alkyl chains was small. However, profiles of the membrane fluidity, which explicitly contain time (expressed as the spin-lattice relaxation rate) and depend on the rotational motion of spin labels, show relatively high fluidity of alkyl chains close to the membrane center. Profiles of the oxygen transport parameter and hydrophobicity have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The amount of CBD was greater in nuclear membranes than in cortical membranes. The presence of the CBD in lens lipid membranes, which at 37 C showed a permeability coefficient for oxygen about 60% smaller than across a water layer of the same thickness, would be expected to raise the barrier for oxygen transport across the fiber cell membrane.