A radical-free preparation of a highly purified ribonucleotide reductase from calf thymus was shown to generate an M2-specific tyrosine free radical on incubation with iron and dithiothreitol in the presence of air. The radical is essential for activity but once formed has a half-life of about 10 min. Using the calf thymus enzyme, there is a continual requirement of oxygen and iron for ribonucleotide reduction indicating a continual regeneration of the radical during enzyme catalysis. We therefore propose that one way a cell may regulate ribonucleotide reductase activity is by controlling the generation of M2-specific tyrosine free radicals within existing M2 molecules.