The thermodynamic parameters, Δ H′, Δ G′, and Δ S′, and the stoichiometry for the binding of the substrate 2′-deoxyuridine-5′-phosphate (dUMP) and the inhibitor 5-fluoro-2′-deoxyuridine-5′-phosphate (FdUMP) to Lactobacillus casei thymidylate synthetase (TSase) have been investigated using both direct calorimetric methods and gel filtration methods. The data obtained show that two ligand binding sites are available but that the binding of the second mole of dUMP is extremely weak. Binding of the first mole of dUMP can best be illustrated by dUMP + TSase + H +⇌(dUMP-TSase-H +). [1] The enthalpy, Δ H 1′, for reaction [1] was measured directly on a flow modification of a Beckman Model 190B microcalorimeter. Experiments in two different buffers ( I = 0.10 m) show that Δ H 1′ = −28 kJ mol −1 and that 0.87 mol of protons enters into the reaction. Analysis of thermal titrations for reaction [1] indicates a free energy change of Δ G 1′ = −30 kJ mol −1 ( K 1 = 1.7 × 10 5 m −1). From these parameters, Δ S 1′ was calculated to be +5 J mol −1 degree −1, showing that the reaction is almost totally driven by enthalpy changes. Gel filtration experiments show that at very high substrate concentrations, binding to a second site can be observed. Gel filtration experiments performed at low ionic strength ( I = 0.05 m) reveal a stronger binding, with Δ G 1′ = −35 kJ mol −1 ( K 1 = 1.2 × 10 6 m −1), suggesting that the forces driving the interaction are, in part, electrostatic. Addition of 2-mercaptoethanol (0.10 m) had the effect of slightly increasing the dUMP binding constant. Binding of FdUMP to TSase is best illustrated by 2FdUMP + TSase + n HH +⇌FdUMP 2 − TSase − (H +) n H . [2] The enthalpy for this reaction, Δ H 2, was also measured calorimetrically and found to be −30 kJ mol −1 with n H = 1.24 at pH 7.4 Assuming two FdUMP binding sites per dimer as established by Galivan et al. [ Biochemistry 15, 356–362 (1976)] our calorimetric results indicate different binding energies for each site. Based on the binding data, a thermodynamic model is presented which serves to rationalize much of the confusing physical and chemical data characterizing thymidylate synthetase.