Hydroxyl radical reacts with the aliphatic C-H bonds of amino acids by H atom abstraction. Under anaerobic conditions inclusion of a H-2 atom donor results in H-1/H-2 exchange into these C-H bonds [Goshe et al. Biochemistry 2000, 39, 1761-1770]. The site of H-1/H-2 exchange can be detected and quantified by H-2 NMR. Integration of the H-2 NMR resonances within a single spectrum permits the relative rate of Ii atom abstraction from each position to be determined. Analysis of the aliphatic amino acid spectra indicates that the methine and methylene positions were more reactive than the methyl positions. The H-2 NMR spectra of isoleucine and leucine show that H-atom abstraction distal to the alpha -carbon occurs preferentially. Significant H-1/H-2 exchange was observed into the delta positions of proline and arginine and into the E-methylene of lysine, indicating that a positive charge on a geminal N does not inhibit the H-1/H-2 exchange. Comparisons of H-2 NMR integrations between amino acid spectra indicated that H-1/H-2 exchange occurred in the following descending order: L > I > V > R > K > Y > P > H > F > M > T > Az [C, S, D, N, E,Q, G, W]. The extent of H-1/H-2 exchange into methionine, N-glycyl-methionine, and methionine sulfoxide suggests that a prominent solvent exchange pathway involving hydroxyl radical mediated oxidation of methionine exists to account for the large H-2 incorporation into the gamma -methylene of methionine sulfoxide that is absent for N-glycyl-methionine. Analysis of the H-1 NMR spectra of the reactions with phenylalanine and tyrosine indicated that hydroxyl radical addition to the phenyl ring under the anaerobic reductive reaction conditions did not result in either exchange or hydroxylation.