The overexpression of P-glycoprotein (Pgp) has been implicated in multidrug-resistance in cancers. Two allosterically-linked drug transport sites have been proposed within Pgp; the H- and R-site, which are selective for Hoechst 33342 (H33342) and rhodamine123, respectively. The allosteric interactions between different Pgp substrates were studied using both ATPase activity and drug transport measurements. A real-time fluorescence assay was used to measure transport of tetramethylrosamine (TMR) and H33342 in proteoliposomes containing purified Pgp. By adding other compounds into the assay (anti-cancer agents, propafenones, peptides), we tested for their inhibition/stimulation of TMR and H33342 transport. Several compounds stimulated the transport of one substrate while inhibiting transport of the other. Kinetic analysis of the initial rate of TMR transport gave Hill coefficient values close to 2, which suggested positive cooperativity. GP03, cyclosporin A, and edelfosine behaved as mixed inhibitors, which suggests that Pgp contains a large substrate binding pocket with multiple binding regions.