Membranes made from three specifically deuterium-labeled ether-linked bolalipids, [1',1',20',20'-H-2(4)[C(20)BAS-PC, [2',2',19',19'-H-2(4)]C(20)BAS-PC, or [10',11'-H-2(2)]C(20)BAS-PC, were analyzed by H-2 NMR spectroscopy. Unlike more common monopolar, ester-linked phospholipids, C(20)BAS-PC exhibits a high degree of orientational order throughout the membrane and the sn-1 chain of the lipid initially penetrates the bilayer at an orientation different from that of the bilayer normal, resulting in inequivalent deuterium atoms at the C1 position. The approximate hydrophobic layer thickness and area per lipid are 18.4 A and 60.4 A(2), respectively, at 25 degrees C, and their respective thermal expansion coefficients are within 20% of the monopolar phospholipid, DLPC.