A 693-base pair gene coding for the 27,132-dalton dihydrofolate reductase (DHFR) domain of the thymidylate synthase-dihydrofolate reductase (TS-DHFR) bifunctional protein of Plasmodium falciparum was designed to have Escherichia coli codon preference and multiple unique restriction sites and was chemically synthesized. The gene was overexpressed (> 50% total cellular protein) in E. coli as insoluble inclusion bodies which could be unfolded and refolded to recover soluble enzyme activity. The refolded DHFR was purified by methotrexate-Sepharose affinity chromatography to give the homogeneous enzyme. Active site titration with methotrexate revealed that the purified protein was fully active. The purified DHFR migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent mass of approximately 30 kDa, and gel filtration showed that the protein is a monomer. The yield of purified enzyme was about 5-6 mg/liter of bacterial culture. Kinetic properties of the purified recombinant DHFR were similar to those reported for wild type bifunctional TS-DHFR. Cassette mutagenesis of the synthetic gene was performed to give the S108N and the N51I + S108N mutants which provided DHFRs analogous to pyrimethamine-resistant mutants found in nature.