The structure of P-glycoprotein (Pgp) has been determined at 3.8 Å resolution, revealing a large internal cavity (~6,000 ų) with a 30 Å separation between the two nucleotide-binding domains (NBDs). The structure shows an inward-facing conformation, which is competent for drug binding. Two additional structures of Pgp with cyclic peptide inhibitors demonstrate distinct drug binding sites in the internal cavity, capable of stereo-selectivity based on hydrophobic and aromatic interactions. The inward-facing conformation allows access to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. Pgp is a major transporter involved in multidrug resistance (MDR) in cancer treatment. It recognizes a wide range of substrates, including hydrophobic compounds, and is often likened to a molecular "hydrophobic vacuum cleaner." The structure of Pgp reveals that the internal cavity can accommodate at least two compounds simultaneously. The binding pocket is composed mainly of hydrophobic and aromatic residues, with only a few polar or charged residues. The co-crystal structures of Pgp with two stereoisomers of cyclic hexapeptide inhibitors, QZ59-RRR and QZ59-SSS, show that Pgp can distinguish between the stereo-isomers, resulting in different binding locations, orientations, and stoichiometry. QZ59-RRR binds to one site per transporter, while QZ59-SSS binds to two sites per Pgp molecule. The binding of these inhibitors is mediated by hydrophobic residues on various transmembrane (TM) helices. The structure of Pgp also shows that the inward-facing conformation is competent for drug binding, and that residues interacting with verapamil are highly conserved, suggesting a common mechanism of poly-specific drug recognition. The drug binding pocket of Pgp is nearly six times larger than that of BmrR and differs significantly from AcrB, where drug binding is mediated by residues from β-sheets on the extracellular side of the inner cell membrane. The inward-facing conformation of Pgp provides access to an internal chamber through two portals, which are open wide enough to accommodate hydrophobic molecules and phospholipids. The structure of Pgp is consistent with previous crosslinking studies and supports the hypothesis that Pgp samples widely open conformations in both detergent-solution and within the membrane to bind larger molecules. The structure of Pgp also suggests that the inward-facing conformation represents a pre-transport state, and that the outward-facing conformation is likely an active state of Pgp.The structure of P-glycoprotein (Pgp) has been determined at 3.8 Å resolution, revealing a large internal cavity (~6,000 ų) with a 30 Å separation between the two nucleotide-binding domains (NBDs). The structure shows an inward-facing conformation, which is competent for drug binding. Two additional structures of Pgp with cyclic peptide inhibitors demonstrate distinct drug binding sites in the internal cavity, capable of stereo-selectivity based on hydrophobic and aromatic interactions. The inward-facing conformation allows access to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. Pgp is a major transporter involved in multidrug resistance (MDR) in cancer treatment. It recognizes a wide range of substrates, including hydrophobic compounds, and is often likened to a molecular "hydrophobic vacuum cleaner." The structure of Pgp reveals that the internal cavity can accommodate at least two compounds simultaneously. The binding pocket is composed mainly of hydrophobic and aromatic residues, with only a few polar or charged residues. The co-crystal structures of Pgp with two stereoisomers of cyclic hexapeptide inhibitors, QZ59-RRR and QZ59-SSS, show that Pgp can distinguish between the stereo-isomers, resulting in different binding locations, orientations, and stoichiometry. QZ59-RRR binds to one site per transporter, while QZ59-SSS binds to two sites per Pgp molecule. The binding of these inhibitors is mediated by hydrophobic residues on various transmembrane (TM) helices. The structure of Pgp also shows that the inward-facing conformation is competent for drug binding, and that residues interacting with verapamil are highly conserved, suggesting a common mechanism of poly-specific drug recognition. The drug binding pocket of Pgp is nearly six times larger than that of BmrR and differs significantly from AcrB, where drug binding is mediated by residues from β-sheets on the extracellular side of the inner cell membrane. The inward-facing conformation of Pgp provides access to an internal chamber through two portals, which are open wide enough to accommodate hydrophobic molecules and phospholipids. The structure of Pgp is consistent with previous crosslinking studies and supports the hypothesis that Pgp samples widely open conformations in both detergent-solution and within the membrane to bind larger molecules. The structure of Pgp also suggests that the inward-facing conformation represents a pre-transport state, and that the outward-facing conformation is likely an active state of Pgp.