Small unilamellar vesicles formed from 1-palmitoyl-2-O-(N-(heptadec-8-cis-enyl)carbamyl)-sn-glycero-3-pho sphocholine (CMPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence of varying amounts of cholesterol have been studied using fluorescence polarization and NMR (1H and 13C) techniques. The fluorescence polarization and 1H-NMR data clearly indicate that the phospholipid packing order in CMPC bilayers is significantly greater than that in the POPC bilayers. The 13C-NMR chemical shift measurements show that this difference between the two phospholipids possibly arises due to the intramolecular hydrogen-bond formation between the -NH and the phosphate residues in the CMPC molecule. It is further shown that unlike POPC, the CMPC packing order is not much affected by including cholesterol in the phospholipid bilayers. These results demonstrate that introduction of one -NH residue adjacent to the C-2 carbonyl carbon in the POPC molecule could make its structure more ordered in the vesicles bilayer, and also would alter its interactions with cholesterol.