Acquired resistance to methotrexate in Leishmania species is often associated with the amplification of H circles, 68 kb duplex DNA circles containing a 30 kb inverted repeat. We report here that the H circle of Leishmania tarentolae contains an open reading frame, ltpgpA, that has the attributes of P-glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). Although amplification of H circles is associated with proportionally increased levels of a 5.5 kb transcript of the ltpgpA gene, such methotrexate resistant mutants are not cross-resistant to any of the drugs extruded by mammalian multi-drug resistant cells. In Leishmania, ltpgpA is part of a gene family containing at least two other members. Sequences homologous to one of the nucleotide binding sites of ltpgpA are conserved in other kinetoplastida.