Microcalorimetry and high-performance liquid chromatography (h.p.l.c.) have been used to conduct a thermodynamic investigation of reactions catalyzed by 3-dehydroquinate synthase and by 3-dehydroquinate dehydratase. These are the second and third reactions in the metabolic pathway leading to the formation of chorismate. The two reactions are: {DAHP(aq)=3-dehydroquinate(aq) + phosphate(aq)} and {3-dehydroquinate(aq)=3-dehydroshikimate(aq) + H 2O(l)}. The h.p.l.c. measurements showed that the first reaction proceeded to completion and that the value of the apparent equilibrium constant for the second reaction was K ′=(4.6±1.5) (Hepes buffer, temperature T=298.15 K, pH=7.50, and ionic strength I m =0.065 mol· kg −1 ). Calorimetric measurements led to a molar enthalpy of reaction Δ r H m ( cal)=−(50.9±1.1) kJ· mol −1 (Hepes buffer, T=298.15 K, pH=7.46, I m =0.070 mol· kg −1 ) for the first reaction and to Δ r H m ( cal)=(2.3±2.3) kJ· mol −1 (Hepes buffer, T=298.15 K, pH=7.42, I m =0.069 mol· kg −1 ) for the second reaction. These results were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at T=298.15 K and I m=0 for chemical reference reactions involving specific ionic forms. For the reaction DAHP 3−(aq)=3-dehydroquinate −(aq)+HPO 4 2−(aq), the standard molar enthalpy of reaction Δ r H° m =−(51.1±4.5) kJ· mol −1 . For the reaction 3-dehydroquinate(aq)=3-dehydroshikimate(aq) +H 2O(l), the equilibrium constant K=(4.6±1.5) and Δ r H° m =(2.3±2.3) kJ· mol −1 . A Benson type approach was used to estimate the standard molar entropy change Δ r S° m for the first reference reaction and led to the value K≈2·10 14 for this reaction. Values of the apparent equilibrium constants and the standard transformed Gibbs free energy changes Δ r G° m under approximately physiological conditions are given for the biochemical reactions.