'H NMR, DSC and UV studies of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) vesicles have demonstrated the favourable physicochemical properties of the transmembrane phospholipid probes 1 a and 1 b for membrane topographical studies. In particular, in the presence of a physiological amount of cholesterol, only one transmembrane conformation is observed. The use of 1 a and cholesterol together for photolabelling experiments in DMPC vesicles led to a re-the myristoyl chains functionalized at markable improvement in the regioselec-C 11, C 12 and C 13 made up 95 % of the tivity of cross-linking between 1 a and total photolabelled myristates, and DMPC, and between 1 a and cholesterol : cholesterol was principally functionalized at the C 25 position on the side-chain. This indicates the formation of a highly ordered bilayer structure and proves directly the orientation of cholesterol perpendicular to the membrane plane with its chain terminal buried in the middle of the bilayer.