The molecular basis of the genetic code manifests itself in the interaction of the aminoacyl-tRNA synthetases and their cognate tRNAs. The fundamental biological question regarding these enzymes' role in the evolution of the genetic code remains open. Here we probe this question in a system in which the same tRNA species is aminoacylated by two unrelated synthetases. Should this tRNA possess major identity elements common to both enzymes, this would favor a scenario where the aminoacyl-tRNA synthetases evolved in the context of preestablished tRNA identity, i.e., after the universal genetic code emerged. An experimental system is provided by the recently discovered O -phosphoseryl-tRNA synthetase (SepRS), which acylates tRNA Cys with phosphoserine (Sep), and the well known cysteinyl-tRNA synthetase, which charges the same tRNA with cysteine. We determined the identity elements of Methanocaldococcus jannaschii tRNA Cys in the aminoacylation reaction for the two Methanococcus maripaludis synthetases SepRS (forming Sep-tRNA Cys ) and cysteinyl-tRNA synthetase (forming Cys-tRNA Cys ). The major elements, the discriminator base and the three anticodon bases, are shared by both tRNA synthetases. An evolutionary analysis of archaeal, bacterial, and eukaryotic tRNA Cys sequences predicted additional SepRS-specific minor identity elements (G37, A47, and A59) and suggested the dominance of vertical inheritance for tRNA Cys from a single common ancestor. Transplantation of the identified identity elements into the Escherichia coli tRNA Gly scaffold endowed facile phosphoserylation activity on the resulting chimera. Thus, tRNA Cys identity is an ancient RNA record that depicts the emergence of the universal genetic code before the evolution of the modern aminoacylation systems.