Isothermal titration calorimetry (ITC) was used to investigate the binding of six inhibitors to 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), a target for developing novel anti-infectives. The binding of hydroxamate inhibitors to E. coli DXR is Mg(2+)-dependent, highly endothermic (ΔH: 22.7-24.3 kJ/mol) and entropy-driven, while that of non-hydroxamate compounds is metal ion independent and exothermic (ΔH: -19.4- -13.8 kJ/mol), showing hydration/dehydration of the enzyme metal ion binding pocket account for the drastic ΔH change. However, for DXRs from Plasmodium falciparum and Mycobacterium tuberculosis, the binding of all inhibitors is exothermic (ΔH: -24.9 - -9.2 kJ/mol), suggesting the metal ion binding sites of these two enzymes are considerably less hydrated. The dissociation constants measured by ITC are well correlated with those obtained by enzyme inhibition assays (R(2) = 0.75). Given the rapid rise of antibiotic resistance, this work is of interest since it provides novel structural implications for rational development of potent DXR inhibitors.