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Drug resistance is a major barrier in the treatment and control of diseases of parasitic origin, but the role of multidrug-resistance (mdr) genes and their products in this drug resistance is equivocal. To determine whether mdr amplification and overexpression can be correlated to a multidrug-resistant (MDR) phenotype in parasitic protozoa, the Idmdrl gene was amplified in L. donovani by direct selection and after transfection. First, Idmdrl was amplified by selecting a mutant strain of L. donovani by virtue of its ability to proliferate in medium containing vinblastine. Second, the ldmdrl copy number was amplified by ligating the gene into an expression plasmid, transfecting the recombinant construct into wild type L. donovani, and selecting for G418 resistance. The phenotype of both strains included cross resistance to multiple structurally and mechanistically unrelated drugs, including vinblastine, puromycin, and the anthracyclines, and an impaired capacity to take up drugs. Nucleotide sequence analysis revealed that Idmdrl encoded a member of the P-glycoprotein (PGP) superfamily that exhibited significant amino acid sequence homology and similar membrane topology to the mammalian mdr PGP. These studies demonstrate that amplification of the ldmdrl by either direct selection or subsequent to transfection can confer a drug resistance phenotype in L. donovani similar to that observed in MDR mammalian cells.