Physicochemical properties of a mixed micellar system of sodium dodecyl sulfate (SDS) and sodium cholate (SC) were investigated. The micelle-micelle and micelle-buffer interactions in an SDS/SC/[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer system for separations by micellar electrokinetic capillary chromatography (MEKC) were studied by capillary electrophoresis (CE) and nuclear magnetic resonance (NMR) techniques. A mixed micellar system of SDS and SC can effectively be used for the separation by MEKC of highly hydrophobic compounds like corticosteroids. Marked differences in distribution coefficients of the corticosteroids were observed when the micellar phase was changed from the highly hydrophobic SDS to the less hydrophobic SC. Measurements of critical micelle concentration were made by CE and conductometric titration. The mixed micellar system was further characterized by NMR methods. NMR-based self-diffusion measurements and NMR relaxation measurements showed that SC affects the micellization of SDS primarily by decreasing the amount of nonmicellized (monomeric) SDS in the mixed system.