The binding of a series of 5-substituted 2'-deoxyuridylates to Lactobacillus casei thymidylate synthetase has been determined by their inhibition of thymidylate formation and of dehalogenation of 5-bromo-2'-deoxyuridylate. As inhibitors of thymidylate formation, the apparent dissociation constants of this series span over three orders of magnitude, and quantitative structure-activity relationships (QSAR) could be established. The most important parameters are the electronic σ- constant and the polar ς constant which indicates that electron withdrawal from the uracil heterocycle increases affinity for the enzyme. The effect cannot be solely ascribed to changes in acidity of the 3-NH and must involve other electronic perturbations of the uracil moiety. The steric parameter, MR, has a negative coefficient indicating that in the series tested the bulkier 5-substituents are detrimental to binding. Hydrophobic effects do not appear to be important in the differential binding of the 5-substituted analogues examined. The same series of analogues shows similar affinities for the enzyme in the absence of the cofactor, as determined by their inhibition of dehalogenation of 5-bromo-2'-deoxyuridylate in the absence of the cofactor, 5,10-methylenetetrahydrofolate. Although QSAR could not be established using this assay, it is clear that the binding regions of 5-substituted 2'-deoxyuridylates are dramatically different in the enzyme and enzyme-cofactor complex. Thymidylate synthetase catalyzes the conversion of 2'-deoxyuridylate (dUMP) to thymidylate (dTMP), with concomitant transfer and reduction of the one carbon unit of 5,10-methylenetetrahydrofolate (CH2-H4folate). Because this enzyme represents the sole de novo pathway for dTMP synthesis, it has been a popular target for design of inhibitors with potential chemotherapeutic applications. Thus far, the most useful inhibitors of thymidylate synthetase have been 5-substituted 2'-deoxyurid ylates. The nature of the 5-substituents has dramatic effects on the affinity for this enzyme, and such analogues seemed ideal candidates for construction of quantitative structure-activity relationships. In this report, we describe equations which correlate the inhibition of Lactobacillus casei thymidylate synthetase by nine 5-substituted 2' deoxyuridylates. The results permit assessment of the features of the 5-substituent which are important for binding and provide a basis for rational development of more potent inhibitors of this enzyme.