The N73 nucleotide at the end of the tRNA acceptor stem is commonly used by tRNA synthetases for discrimination. Because only a few synthetase-tRNA cocrystal structures have been determined, understanding of the molecular basis for N73 discrimination is limited. Here we investigated the possibility that, for at least some synthetases, the capacity to recognize different N73 nucleotides resides in the variable sequence of the loop of motif 2, a motif found in all class II enzymes. In the cocrystal of the class II yeast aspartyl-tRNA synthetase, atomic groups of the G73 discriminator of tRNAAsp interact with three side chains of the enzyme. We examined lysyl-tRNA synthetase, a close structural homologue of the aspartyl enzyme. Different substitutions were introduced into the Escherichia coli enzyme (A73 discriminator) to make its loop more like that of the human enzyme (G73 discriminator). Our data show that the loop of motif 2 of the lysine enzyme makes tRNA functional contacts, as predicted from the structural comparison. And yet, the E. coli enzyme with the "humanized" loop sequence had the same quantitative kinetic preference for A73 versus G as the wild-type enzyme. We conclude that discriminator base selectivity in the lysine enzyme requires residues in addition to or other than those in the loop of motif 2. Thus, even tRNA synthetases that are close structural homologues may use the same RNA binding element to make functional contacts with places (in the acceptor stem) that are idiosyncratic to each synthetase-tRNA pair.