A general and relatively simple method for the acylation of aminoacyl-tRNA is described. The synthesis involves a reaction between N- hydroxysuccinimide esters of short- and long-chain fatty acids and aminoacyl-tRNA. The activated fatty acids react specifically with the amino acid attached to the tRNA and do not react with the tRNA proper. The acylated phenylalanyl-tRNA did not lose its biological properties as a result of the acylation reaction. Acylphenylalanyl-tRNA could be bound to ribosomes in a poly (U)-dependent reaction. The tRNA could be recharged with phenylalanine after removal of the acylphenylalanine from the tRNA by mild alkaline hydrolysis.