Solute partitioning into vesicles of dihexadecyl hydrogen phosphate (DHP) was investigated by electrokinetic chromatography (EKC). The ionic double-chain surfactant forms bilayer structures that mimic a biological membrane when dispersed in aqueous media. The linear solvation energy relationship (LSER) was used to gain insights about the nature of interactions and parameters that influence the partitioning process into vesicles, micelles, and n-octanol. The Gibbs free energies of transfer of selected functional groups from aqueous to vesicular phases were compared to those in micellar pseudophases of sodium dodecyl sulfate (SDS) and sodium dodecyl phosphate (SDP). Size and hydrogen bond acceptor strength of solutes are the main factors that determine their partitioning behavior, while dipolarity and polarizability play minor, yet significant, roles. It was determined that the cohesiveness of solute microenvironments in DHP vesicles is between those for micells and n-octanol. Vesicles are weaker hydrogen bond donors and less dipolar than micelles; however, they interact more strongly with the n and pi electrons of solutes. Interestingly, as the organization of the pseudophase is enhanced from micelles to vesicles, their microenvironments become closer to that of octanol. Consequently, solute partitioning in vesicle-water correlates better with that in octanol-water than with that in micelle-water. The influence of the presence of cholesterol in the bilayer on the interactive nature of the pseudophase was also investigated.