Transfer RNAs terminating 2'-or 3'-deoxyadenosine were prepared from unfractionated E. coli and yeast (Saccharomyces cerevisiae) tRNAs and purified to remove unmodified tRNAs. The modified tRNA species were assayed for aminoacylation with each of the 20 amino acids to determine the initial position of tRNA aminoacylation. The E. coli and yeast aminoacyl-tRNA synthetases specific for arginine, isoleucine, leucine, methionine, phenylalanine, and valine, as well as the E. coli glutamyl-tRNA synthetase, aminoacylated only those cognate tRNAs terminating in 3'-deoxyadenosine (i.e., those having a 2'-OH group). On the other hand, those E. coli and yeast synthetases specific for alanine, glycine, histidine, lysine, proline, serine, and threonine, as well as the yeast synthetase specific for glutamine, utilized exclusively those tRNAs having an available 3'-OH group on the 3'-terminal nucleoside, while the E. coli and yeast synthetases specific for asparagine, cysteine, and tyrosine, and the yeast aspartyl-tRNA synthetase, utilized both of the modified cognate tRNAs. The only observed difference in specificity between the E. coli and yeast systems was for tRNATrp, which was aminoacylated on the 2'-position in E. coli and the 3'-position in yeast. The results indicate that the initial position of aminoacylation is not uniform for all tRNAs, although for individual tRNAs the specificity has been conserved during the evolution from a prokaryotic to eukaryotic organism.