Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA Gln is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA Glu and tRNA Gln with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA Glu. Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS Tel ) in complex with glutamate at a resolution of 2.45 Å. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS Tth ). We confirm experimentally that GluRS Tel is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA Glu and of Glu-tRNA Gln. Anticodons of tRNA Glu ( 34C/UUC 36) and tRNA Gln ( 34C/UUG 36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS Tth by the residue Arg358. In ND-GluRS Tel this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA Gln to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.