Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization.