Thymidylate synthase catalyzes the reductive methylation of dUMP to dTMP and is essential for the synthesis of DNA. Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluoro-2'-deoxyuridine 5'-monophosphate, a tight binding covalent inhibitor of thymidylate synthase. Recent studies have identified 5-fluoro-2'-deoxyuridine (5-FdUR) and antifolate-resistant mutants of human thymidylate synthase (TS) that contain single residue substitutions within the highly conserved Arg50-loop, which binds the pyrimidine substrate (Y. Tong et al., J. Biol. Chem. 273: 11611-11618, 1998). We have used random sequence mutagenesis to gain structure-function information about the TS and to create novel drug-resistant mutants for gene therapy. A library of 1.5 million mutants of the Arg50-loop and the nearby residue Tyr 33 was selected to identify mutants of the human enzyme with the ability to complement a thymidylate synthase-deficient Escherichia coli strain and form colonies in the presence of 5-FdUR. E. coli-harboring plasmids that were encoding TS with single, double, and triple amino acid substitutions were identified that survive at dosages of 5-FdUR clearly lethal to E. coli harboring either wild-type thymidylate synthase or constructs encoding previously characterized drug resistant mutants. Four 5-FdUR-resistant mutants were purified to apparent homogeneity. Kinetic studies indicate that these enzymes are highly efficient. Inhibition constants (Ki) for the double mutant K47Q;D48E and the triple mutant D48E;T51S;G52C in the presence of 5-fluoro-2'-deoxyuridine 5'-monophosphate were determined to be 75 to 100 times higher, respectively, than that of the wild-type enzyme. These mutant TSs, or others similarly created and selected, could be used to protect bone marrow cells from the cytotoxic side effects of 5-FU chemotherapy.