The recognition of transfer RNAs (tRNAs) by aminoacyl tRNA synthetases is a critical step in establishing the fidelity of translation. For E. coli cysteine tRNA synthetase, recognition of tRNA(Cys) and discrimination from all other tRNAs is based on the U73 discriminator base, the GCA anticodon, and a G15:G48 tertiary base pair. While the discriminator base and the anticodon sequence are often used by many synthetases as the determinants for tRNA recognition, the dependence on a tertiary interaction in the cognate tRNA for recognition is unique to the cysteine enzyme. Here the structural basis for recognition at the G15:G48 tertiary interaction of E. coli tRNA(Cys) is explored by structural modeling, chemical modifications, and kinetic analysis. The results established an unusual RNA tertiary interaction that provides a plausible mechanism for recognition by cysteine tRNA synthetase.