A liquid chromatographic stationary phase containing immobilized P-glycoprotein (Pgp) was synthesized using cell membranes obtained from Pgp-expressing cells. The resulting Pgp-stationary phase was used in frontal and zonal chromatographic studies to investigate the binding of vinblastine (VBL), doxorubicin (DOX), verapamil (VER), and cyclosporin A (CsA) to the immobilized Pgp. The compounds were added individually to the chromatographic system with or without ATP in the running buffer. Using this approach, dissociation constants were calculated for VBL (23.5 ± 7.8 nM), DOX (15.0 ± 3.2 μM), VER (54.2 ± 4.7 μM), and CsA [97.9± 19.4 nM (without ATP) and 62.5 ± 4.6 nM (with ATP)]. The compounds were also added in pairs using standard competitive chromatography procedures. The results of the study demonstrate that competitive interactions occurred between VBL and DOX, cooperative allosteric interactions occurred between VBL and CsA and ATP and CsA, and anticooperative allosteric interactions occurred between ATP and VBL and VER. The chromatographic studies indicate that the immobilized Pgp was modified by ligand and cofactor binding and that the stationary phase can be used to study drug-drug binding interactions on the Pgp molecule.