As reported earlier, the marine sponge phospholipids 1,2-di-5,9-hexacosadienoyl-sn-glycero-3-phosphocholine (PC (26 : 2, 26 : 2)) and its phosphoethanolamine counterpart exclude cholesterol to a great extent from their liposomal bilayers. To understand the reason for this exclusion, shorter acyl chain analogs of these phospholipids were synthesized. Liposomes composed of PC (18:2, 18 : 2), PC (22 : 2, 22 : 2) or PC (24: 2, 24 : 2), with the unique A ~,9 unsaturation typical of sponge phospholipids, incorporated cholesterol. As.9-pE (24 : 2, 24 : 2) which also incorporated cholesterol, had a phase transition temperature identical to its PC counterpart. Differential scanning calorimetry indicated that •5.9-pc (26:2, 26:2) could incorporate l-stearoyl-2-oleoyl-sn-glycerophosphocholine PC (18 : 0, 18 : 1) into its bilayers at various ratios (20, 40, 60, 80 mol%), but part of PC (18 : 0, 18 : 1) displayed monotectic behavior. When 20 mol cholesterol was added to these mixtures, the sterol interacted preferentially with the PC (18 : 0, 18 : l) but was also incorporated in all ratios into the mixture of phospholipids and created one major transition endotherm.