Surface pressure-area isotherm, neutron specular reflection, and small-angle neutron scattering studies have been carried out to determine the effects of added cholesterol and distearoylphosphatidylcholine (DSPC), on the molecular structures of monolayers and vesicles containing the dialkyl polyoxyethylene ether surfactant, 1,2-di-O-octadecyl-rac-glyceryl-3-(omega-dodecaethylene glycol) (2C(18)E(12)). Previous neutron reflectivity studies on 2C(18)E(12) monolayers at the air/water interface have shown them to possess a thickness of similar to24 Angstrom and highly disordered structure with significant intermixing of the polymer headgroups and alkyl chains. SANS studies of 2C(18)E(12) vesicles gave a bilayer thickness of similar to51 Angstrom. Addition of cholesterol to 2C(18)E(12) monolayers (1:1 molar ratio), produced a marked condensing effect coupled with an increased the layer thickness of similar to7 Angstrom, and in vesicles, increased bilayer thickness by similar to16 Angstrom. Monolayers consisting of 2C(18)E(12):DSPC:cholesterol (1:1:2 molar ratio), showed a layer thickness of similar to31 Angstrom, whereas in vesicles, three-component bilayer was found to be only similar to9 Angstrom thicker than those possessed by vesicles composed solely of 2C(18)E(12). Mixing between the molecules in three-component monolayers was shown to be ideal through analysis of the neutron reflectivity data. These findings are discussed in relation to increased ordering and decreased headgroup/hydrophobe intermixing within both monolayers and vesicle bilayers containing 2C(18)E(12). The inferred increase in molecular order within vesicles composed of 2C(18)E(12) with additional cholesterol and phospholipid is used as a model for explaining theoretical differences in bilayer permeability.