The interaction of Lactobacillus casei thymidylate synthase (TS) with 10-propargyl-5,8-dideazafolate (NPQ) in the presence of 2'-deoxyuridylate (dUMP) has been investigated. After formation of a rapidly reversible dUMP-NPQ-enzyme complex, a slow isomerization occurs to provide a ternary complex that can be isolated on nitrocellulose membranes or by gel filtration. Unusual features of the isolable complex are the slow rate by which it is formed (t1/2 = 0.88 h) and the slow rate at which it dissociates (t1/2 = 26.5 h). The ternary complexes contain 2 mol of dUMP and 2 mol of NPQ bound per mol of dimeric enzyme. Ultraviolet difference spectra of the dUMP-NPQ-TS complex shows a high wavelength maximum that has been attributed to perturbations of the enzyme and/or ligand chromophores that occur upon binding. Data are presented that suggest that the formation of the isolable ternary complex involves nucleophilic attack by a catalytic thiol group of the enzyme to the 6-position of dUMP. Evidence for this is as follows: first, there is a decrease in the absorbance of the pyrimidine chromophore at 265 nm that occurs at the same rate as the formation of the isolable complex; second, using [6-3H]dUMP there is a large, inverse alpha-secondary kinetic isotope effect (kappa H/kappa T = 0.83) upon formation of the complex that is in accord with sp2 to sp3 rehybridization of the 6-carbon of the heterocycle. Treatment of the complex with sodium dodecyl sulfate (NaDodSO4) results in the dissociation of both ligands in an unmodified form, which is consistent with proposed structure of the complex. Isolable ternary complexes are also formed when the enzyme is incubated with 5-fluoro-2'-deoxyuridylate (FdUMP) and NPQ. Interestingly, the dissociation of FdUMP from these complexes is biphasic, with one-half of the bound nucleotide dissociating at an exceedingly slow rate (t1/2 congruent to 100 h). The findings are discussed with relationship to the possible use of NPQ as an anticancer agent.