Thymidylate synthase (TS) from Lactobacillus casei is inhibited by 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF3dUMP). CF3dUMP binds to the active site of TS in the absence of 5,10-methylenetetrahydrofolate, and attack of the catalytic nucleophile cysteine 198 at C6 of the pyrimidine leads to activation of the trifluoromethyl group and release of fluoride ion. Subsequently, the activated heterocycle reacts with a nucleophile of the enzyme to form a moderately stable covalent complex. Proteolytic digestion of TS treated with [2'-3H]CF3dUMP, followed by sequencing of the labeled peptides, revealed that tyrosine 146 and cysteine 198 are covalently bound to the inhibitor in the enzyme-inhibitor complex. The presence of dithiothreitol (DTT) or beta-mercaptoethanol resulted in the breakdown of the covalent complex, and products from the breakdown of the complex were isolated and characterized. The three-dimensional structure of the enzyme-inhibitor complex was determined by X-ray crystallography, clearly demonstrating covalent attachment of the nucleotide to tyrosine 146. A chemical reaction mechanism for the inhibition of TS by CF3dUMP is presented that is consistent with the kinetic, biochemical, and structural results.