Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl–tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNAArg can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNAArg isoacceptors and heterologous tRNAAsp, we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination.