Since the discovery of the essential tyrosyl radical (Y*) in E. coli ribonucleotide reductase (RNR), a number of enzymes involved in primary metabolism have been found that use transient or stable tyrosyl (Y) or tryptophanyl (W) radicals in catalysis. These enzymes engage in a myriad of charge transfer reactions that occur with exquisite control and specificity. The unavailability of natural amino acids that can perturb the reduction potential and/or protonation states of redox-active Y or W residues has limited the usefulness of site-directed mutagenesis methods to probe the attendant mechanism of charge transport at these residues. However, recent technologies designed to site-specifically incorporate unnatural amino acids into proteins have now made viable the study of these mechanisms. The class Ia RNR from E. coli serves as a paradigm for enzymes that use amino acid radicals in catalysis. It catalyzes the conversion of nucleotides to deoxynucleotides and utilizes both stable and transient protein radicals. This reaction requires radical transfer from a stable tyrosyl radical (Y(122)*) in the beta subunit to an active-site cysteine (C(439)) in the alpha subunit, where nucleotide reduction occurs. The distance between the sites is proposed to be >35 A. A pathway between these sites has been proposed in which transient aromatic amino acid radicals mediate radical transport. To examine the pathway for radical propagation as well as requirements for coupled electron and proton transfers, a suppressor tRNA/aminoacyl-tRNA synthetase (RS) pair has been evolved that allows for site-specific incorporation of 3-aminotyrosine (NH(2)Y). NH(2)Y was chosen because it is structurally similar to Y with a similar phenolic pK(a). However, at pH 7, it is more easily oxidized than Y by 190 mV (approximately 4.4 kcal/mol), thus allowing it to act as a radical trap. Here we present the detailed procedures involved in evolving an NH(2)Y-specific RS, assessing its efficiency in NH(2)Y insertion, generating RNR mutants with NH(2)Y at selected sites, and determining the spectroscopic properties of NH(2)Y* and the kinetics of its formation.