Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in the trypanosomatids. We report here the cloning by expression of the Leishmania donovani gene. It is single copy, expresses a 2.6-kb transcript and a 52-kDa protein and is located on a 1.1-Mbp chromosome. The 491 amino acid sequence has 76% similarity to Crithidia fasciculata and 67% similarity to Trypanosoma cruzi, Trypanosoma congolense and Trypanosoma brucei TR. Residues recognising the adenosine pyrophosphate moiety of NADPH and FAD, and residues in the catalytic site segment (A47-A67) involving electron transfer from TR to trypanothione disulphide (T(S)2) were completely conserved. Thus inhibitors of TR are likely to be active against the enzyme from all the parasitic trypanosomatids. Two peptide inserts (39-47, 131-140) seen in other TR genes and a C-terminal extension of 19 residues were also present. When the gene was introduced back into L. donovani at high copy number using the pTEX expression vector, we detected elevated expression of TR RNA and a 14-fold increase in TR activity. Transfection and overexpression of the TR gene will facilitate studies of gene function and of the dependence of trypanosomatids on TR for protection against oxidative stress.