A tRNA with "double identity" was created, and this tRNA was demonstrated in vitro to aminoacylate quantitatively with either of two amino acids. In contrast, acceptance of only one of these amino acids was observed in vivo, and a simple manipulation determined which one was accepted. Kinetic parameters were obtained for aminoacylation with each amino acid of the tRNA with double identity and of related tRNAs. Modeling with these parameters largely explains which amino acid specificity is observed in vivo. The results delineate some of the kinetic boundaries for the design and accommodation of tRNA sequence variations in the elaboration of identity in vivo.