Early biochemical data showed that aminoacyl-tRNA synthetases often displayed species-specific recognition of tRNA. We compared the ability of purified Saccharomyces cerevisiae and Escherichia coli arginyl-tRNA synthetases to aminoacylate native and transcribed yeast tRNA A r g as well as E. coli tRNA A r g . The kinetic data revealed that yeast ArgRS could charge E. coli tRNA A r g , but at a lower efficiency than it charged either the transcribed or native yeast tRNA A r g . E. coli ArgRS can acylate only its cognate E. coli tRNA. Strikingly, a single base change from C to A at position 20 in yeast tRNA 3 A r g altered the species specificity. The transcript of yeast tRNA 3 A r g CA20 mutant was aminoacylated by E. coli ArgRS with a 10 6 increase in k c a t /K m over that for aminoacylation of yeast tRNA 3 A r g transcript. This indicates that A20 is not only an important identity of E. coli tRNA A r g , but is also the key to altering species-specific aminoacylation of yeast tRNA A r g .