Volume changes associated with the partitioning of eight different alcohols and acetone into unilamellar vesicles of dimyristoyl phosphatidylcholine in the fluid state was investigated by newly designed, automated titration densitometric equipment. The change in free volume of the partitioning process, expressed by changes in the specific excess volume of the solutions, was positive for all solutes. The magnitude of the total volume change per mole alcohol partitioned, DeltaV(m), was calculated by combining the volumetric data with previously published partitioning coefficients. For aliphatic 1-alcohols, DeltaV(m) increased gradually from 3 cm(3)/mol for ethanol to similar to 29 cm(3)/mol for 1-hexanol. Values of DeltaV(m) for the cyclic alcohols c-hexanol and benzyl alcohol were similar to3 times smaller than for aliphatic compounds of a similar molecular weight. The composition dependence of the volumetric excess functions could be determined with sufficient accuracy to allow estimation of the partial specific excess volume of all three components (water, alcohol, and lipid). These functions elucidate the contribution of each component to the measured expansion of the system. The partial volume of vesicles and alcohols increased, whereas the volume of water was diminished as a result of the partitioning process. This result suggests that the volume increase of the membrane-alcohol complex is larger than the measured volume change of the whole system (DeltaV(m)) because the latter is partially compensated by contraction of the solvent. The results support a molecular picture in which adsorption of alcohols promotes a loosening of the molecular packing and an increase in the water content of lipid bilayer membranes.