P-glycoprotein (Pgp) is an ABC superfamily protein that exports lipid-soluble substrates from the cell using the energy of ATP hydrolysis. Pgp has been implicated as a major factor in multidrug resistance of human cancers. Pgp is proposed to operate as a vacuum cleaner, transporting substrates from within the bilayer. A series of rhodamine dyes was characterized with respect to their affinity for binding to purified Pgp reconstituted into proteoliposomes, at temperatures above and below the lipid melting transition. Pgp binding affinity (Kd) was determined using a fluorescence-based Trp quenching assay, and the lipid-water partition coefficient (Plip) was determined using equilibrium dialysis. There was a strong correlation between binding affinity and Plip for the rhodamine series. For bilayers of dimyristoylphosphatidylcholine, Plip increased and Pgp binding affinity decreased as the temperature was increased through the bilayer melting transition.