Abstract—The photosensitized oxygenation of cholesterol in liposomal membranes results in the formation of the characteristic singlet oxygen product, 3β‐hydroxy‐5α‐cholest‐6‐ene‐5‐hydroperoxide. The yield of this product was found to be strongly temperature dependent with 6‐ to 7‐fold increase above the transition temperature of the membrane using 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphorylcholine or 1.2‐dimyristoyl‐sn‐glycero‐3‐phosphorylcholine. In contrast, the formation of the radical autoxidation products of cholesterol was not significantly enhanced in these systems by higher temperatures. We conclude from the results that the oxidation of cholesterol by singlet oxygen in the artificial membranes is affected by both membrane fluidity and amount of hematoporphyrin incorporated into the membrane. The ratio of cholesterol to phospholipid which affects the morphology of membranes is a crucial factor in determinin