Pteridine reductase I (PTR I) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR I is involved opens the possibility of developing specific inhibitors, which in combination with dihydrofolate reductase inhibitors could be highly effective against Leishmania. In order to increase our understanding of this putative important chemotherapeutic target, we present here the cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India. This recombinant enzyme will set the basis for inhibition studies as well as for structure-function relationships. (C) 2004 Elsevier Inc. All rights reserved.