One cause of multidrug resistance is the overexpression of P-glycoprotein, a 170 kDa plasma membrane ABC transporter, which functions as an ATP-driven efflux pump with broad specificity for hydrophobic drugs, peptides, and natural products. The protein appears to interact with its substrates within the membrane environment. Previous reports suggested the existence of at least two binding sites, possibly overlapping and displaying positively cooperative interactions, termed the H and R sites for their preference for Hoechst 33342 and rhodamine 123, respectively. In this work, we have used several fluorescence approaches to characterize the molecular interaction of purified P-glycoprotein (Pgp) with the dye LDS-751, which is proposed to bind to the R site. A 50-fold enhancement of LDS-751 fluorescence indicated that the protein binding site was located in a hydrophobic environment, with a polarity lower than that of chloroform. LDS-751 bound with sub-micromolar affinity (K(d) = 0.75 microM) and quenched P-glycoprotein intrinsic Trp fluorescence by 40%, suggesting that Trp emitters are probably located close to the drub-binding regions of the transporter and may interact directly with the dye. Using a FRET approach, we mapped the possible locations of the LDS-751 binding site relative to the NB domain active sites. The R site appeared to be positioned close to the membrane boundary of the cytoplasmic leaflet. The location of both H and R drug binding sites is in agreement with the idea that Pgp may operate as a drug flippase, moving substrates from the inner leaflet to the outer leaflet of the plasma membrane.