The lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) forms a lamellar liquid crystalline phase (L alpha) in arbitrary mixtures of glycerol and water. The phase has been characterized by means of X-ray diffraction, 31P-NMR spectroscopy and differential scanning calorimetry (DSC). In the L alpha state, and for DOPC concentrations greater than 50% (w/w), the thickness of the lipid bilayer decreases, while the area of the polar head group increases with increasing glycerol concentration. The phase transition from gel to L alpha state occurs in the range of 240 to 260 K. Contrary to a previous (McDaniel, R.V., McIntosh, T.J. and Simon, S.A. (1983) Biochim. Biophys. Acta 731, 97) study of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) we find that in the gel state, the thickness of the DOPC lipid bilayer is greater than that in the L alpha state. This suggests that in the gel state, the lipid acyl chains of DOPC are in extended configuration. The lamellar phase reaches its maximum swelling at about 50% (w/w) of DOPC. At lower DOPC concentrations a two-phase system is formed where the lamellar phase exists in equilibrium with excess of solvent. Unilamellar vesicles can be prepared from a diluted suspension of the lamellar phase either by using the sonicator or extruder technique. We show this by means of 31P-NMR, EPR and fluorescence spectroscopy. The mean radius of the vesicles, prepared by a sonicator, has been determined at different glycerol/water mixtures. It is found to decrease continuously from 100 A at 100% water to a minimum of 75 A at about 50% water in the solvent mixture. By further decreasing the water content in the solution, the radius rapidly increases, and a mean radius of 450 A is estimated at a water content of 10%. The rotational relaxation times of a fluorescent probe and two EPR spin probes, solubilized in DOPC vesicles, have been measured at different glycerol/water mixtures. It is found that the rotational rates are always much slower in the systems containing glycerol.