To prevent mistranslation, aminoacyl-tRNA synthetases (AARSs) discriminate against noncognate amino acids and cellular metabolites. Defects in specificity produce statistical proteins which, in mammalian cells, lead to activation of the unfolded protein response and cell death. Because of inherent limitations in amino acid discrimination by a single active site, AARSs evolved a separate domain to clear mischarged amino acids. Although the structure of a widely distributed editing domain for ThrRS and AlaRS is known, the mechanism of amino acid clearance remains elusive. This domain has two motifs that together have four conserved residues in the pocket used to clear serine from mischarged tRNAs. Here, using ThrRS as an example, rapid single-turnover kinetics, mutagenesis, and solvent isotope analysis show that a strictly conserved histidine (between ThrRS and AlaRS) extracts a proton in the chemical step of the editing reaction. Three other conserved residues, and two additional residues in the editing pocket, are not directly implicated in the chemical step. These results are relevant to the previously reported mutagenesis of the homologous editing pocket of alanyl-tRNA synthetase, where even a mild defect in editing causes neurodegeneration in the mouse. Thus, a single proton-transfer event needed to prevent mistranslation can have profound implications for disease.