The R2 protein of ribonucleotide reductase contains at the side chain of tyrosine 122 a stable free radical, which is essential for enzyme catalysis. The tyrosyl radical is buried in the protein matrix close to a dinuclear iron center and a cluster of three hydrophobic residues (Phe-208, Phe-212, and Ile-234) conserved throughout the R2 family. A key step in the generation of the tyrosyl radical is the activation of molecular oxygen at the iron center. It has been suggested that the hydrophobic cluster provides an inert binding pocket for molecular oxygen bound to the iron center and that it may play a role in directing the oxidative power of a highly reactive intermediate toward tyrosine 122. We have tested these hypotheses by constructing the following mutant R2 proteins: F208Y, F212Y, F212W, and I234N. The resulting mutant proteins all have the ability to form a tyrosine radical, which indicates that binding of molecular oxygen can occur. In the case of F208Y, the yield of tyrosyl radical is substantially lower than in the wild-type case. A competing reaction resulting in hydroxylation of Tyr-208 implies that the phenylalanine at position 208 may influence the choice of target for electron abstraction. The most prominent result is that all mutant proteins show impaired radical half-life; in three of the four mutants, the half- lives are several orders of magnitude shorter than that of the wild-type radical. This suggests that the major role of the hydrophobic pocket is to stabilize the tyrosyl radical. This hypothesis is corroborated by comparative studies of the environment of other naturally occurring tyrosyl radicals.