Glutamate 60 of thymidylate synthase coordinates a hydrogen bond network important in proton transfer reactions to and from the substrate dUMP. The E60A and E60L mutants of Lactobacillus casei thymidylate synthase catalyzed tritium exchange from [5-3H]dUMP for solvent protons faster than dTMP formation, indicating accumulation of a steady-state intermediate and a change in partitioning of the intermediate. A covalent complex consisting of E60A or E60L thymidylate synthase, dUMP, and the cofactor CH2H4 folate was isolated on SDS-polyacrylamide gel electrophoresis and shown to be chemically and kinetically competent to form dTMP. These results provide proof of the formation of a covalent steady-state intermediate in the reaction pathway of thymidylate synthase and demonstrate that the rate-determining step in the mutants occurs during conversion of the covalent intermediate to dTMP.