Three enzyme activities in the protozoan Leishmania major, namely N-5,N-10-methylenetetrahydrofolate dehydrogenase/N-5,N-10-methenyltetrahydrofolate cyclohydrolase (DHCH) and N-10-formyltetrahydrofolate ligase (FTL) produce the essential intermediate N-10-formyltetrahydrofolate. Although trypanosomatids possess at least one functional DHCH, the same is not true for FTL, which is absent in Trypanosoma brucei. Here, we present the 2.7 angstrom resolution crystal structure of the bifunctional apo-DHCH from L major, which is a potential drug target. Sequence alignments show that the cytosolic enzymes found in trypanosomatids share a high level of identity of approximately 60%. Additionally, residues that interact and participate in catalysis in the human homologue are conserved amongst trypanosomatid sequences and this may complicate attempts to derive potent, parasite specific DHCH inhibitors. (C) 2011 Elsevier B.V. All rights reserved.