The ordering properties of sn-1 and sn-2 dimyristoylphosphatidylcholine (DMPC) chains are investigated by NMR and neutron diffraction in order to relate membrane defects to bilayer thickness. First, the effect of temperature and 30 mol % cholesterol on DMPC membranes perdeuterated on the sn-1 acyl chain is studied by solid state H-2-NMR. Using the concept of C-C bond order parameters, S-CC, a comparison is made between both lipid chains according to our previously published data on sn-2 chains (Douliez; et al. Biophys. J. 1995, 68, 1727-1739). An odd-even effect is observed when plotting S-CC versus carbon position, k, the amplitude of which is constant when increasing temperature, in the presence or absence of cholesterol. Such an effect can be related to different dynamics of conformers used to describe C-C bond isomerization. It is also found that S-odd(CC) < S-even(CC) for sn-1 chains and S-odd(CC) > S-even(CC) for sn-2 chains. Such an inversion in parity is accounted for by the peculiar conformational dynamics occurring around C-1-C-2 in the sn-2 chain. Calculation of the carbonyl C-1-C-2 bond order parameter indicates that the beginning of the sn-1 chain tends to be perpendicular to the plane of the bilayer, i.e. conversely to what has been found for the C-1-C-2 bond of the sn-2 chain which orients parallel to the bilayer surface. The presence of cholesterol does not markedly modify the dynamics of the C-1-C-2 bond of the sn-2 chain, whereas it does restrict by 70% the conformational freedom of the beginning of the sn-1 chain. The number of gauche defects for the sn-1 chain increases from 3 to 4.3 on going from 25 to 60 degrees C and is greater by 0.5-0.7 unit than that of the sn-2 chain, at corresponding temperatures. The average length of the sn-1 chain is found to be 12.7 Angstrom at 25 degrees C and 11.0 Angstrom at 50 degrees C, i.e. it is greater than that of the sn-2 chain by 0.7 Angstrom close to the main transition temperature, T-C, whereas both chains have the same length for T > T-C + 15 degrees C. At corresponding temperatures, addition of cholesterol results in a 3.5 Angstrom increase in the sn-1 chain length. Second, measurement of the peak-to-peak distance in the one-dimensional neutron-scattering amplitude density of the membrane leads to a bilayer hydrophobic thickness, d(h), of 29.3 and 33.2 Angstrom at 50 degrees C, in the absence and presence of 30 mol % cholesterol, indicating a large apparent discrepancy between d(h) and twice the acyl chain length as calculated from NMR. However, comparison of chain length determination by NMR and neutron diffraction using deuterium labels at positions 5,15 and 4,14 on one of the dipalmitoylphosphatidylcholine acyl chains shows that both methods measure the same quantity within 0.4 Angstrom. Hence, the marked differences observed between neutron diffraction and solid state NMR in bilayer hydrophobic thickness determination are interpreted in terms of protrusion of molecules above the average bilayer surface. This protrusion is estimated to be 2.1 Angstrom for DMPC at 50 degrees C and is reduced to 0.7 Angstrom in the presence of 30 mol % cholesterol.