A new chromatographic method was developed for characterizing allosteric interactions between an immobilized binding agent and low-solubility compounds. This approach was illustrated by using it to characterize the interactions between tamoxifen and warfarin during their binding to the protein human serum albumin (HSA), with beta-cyclodextrin being employed as a solubilizing agent for these drugs. It was confirmed in this work through several experiments that warfarin had a single binding site on HSA with an association equilibrium constant of (2-5) x 10(5) M(-1) (average, 3.9 x 10(5) M(-1)) at 37 degrees C, in agreement with previous reports. It was also found that tamoxifen had a single major binding site on HSA, with an association equilibrium constant of (3-4) x 10(7) M(-1) (average, 3.5 x 10(7) M(-1)) at 37 degrees C. When warfarin was used as a mobile-phase additive in competition studies with tamoxifen, this had a positive allosteric effect on tamoxifen/HSA binding, giving a coupling constant of 2.3 (+/-0.3). Competitive studies using tamoxifen as a mobile-phase additive indicated that tamoxifen had a negative allosteric effect on warfarin/HSA binding, providing a coupling constant of 0.79 (+/-0.03). A unique feature of the technique described in this report was its ability to independently examine both directions of the warfarin/tamoxifen allosteric interaction. This approach is not limited to warfarin, tamoxifen, and HSA but can also be used to study other solutes and binding agents.