The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase was engineered to contain a prokaryotic ribosome binding site and was expressed under the bacteriophage T7 RNA polymerase promoter in Escherichia coli. Site-directed mutagenesis was used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase (D/C) domain as amino acid residues 1-301, and the synthetase (Syn) domain as residues 304-935. Both domains formed active enzymes thereby demonstrating their ability to fold independently. The full-length enzyme, D/C and Syn domains were expressed at levels 4-, 55- and 3-fold higher than the specific activities found in liver. Additional mutagenesis and independent expression of domains further defined the interdomain region to include amino acids 292-310. The D/C domain was purified to homogeneity by a single affinity chromatographic step, and the full-length protein in a two-step procedure. The kinetic properties of the D/C domain appear unaltered from those of the trifunctional enzyme.