The interaction of estrone and estradiol with b-cyclodextrins (bCD) was investigated by differential pulse voltammetry (DPV) and high-performance liquid chromatography (HPLC) in mixed media. The co-solvent influence on the tendency of these estrogens to form inclusion complexes with bCD was examined. Thus, acetonitrile (MeCN) and ethanol (EtOH) were used in a mixed aqueous medium containing phosphate buffer. The association constant of the inclusion complexes (K a ) of estrone and estradiol with bCD were determined in two different media by using both voltammetric and chromatographic techniques. Estradiol was found to bind to bCD with higher affinities than estrone, irrespective of the medium. We have also found a clear influence of the co-solvent on the K a value, which means a competition of co-solvent molecules with estrogens for binding to the cavity of bCD. Consequently, interaction between bCD and the steroids is weakened when acetonitrile is used.