Ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, the latter of which contains an iron-tyrosyl free radical center essential for activity. We have studied the in vivo effects on the R2 protein of the potent iron chelators parabactin and desferrioxamine using R2-overproducing mouse cells with a tyrosyl free radical signal easily quantifiable by EPR spectroscopy. Both chelators inhibited cell growth, and the inhibition was reversible by iron. Furthermore, both chelators, which penetrate cells and chelate the intracellular iron pool, caused a disappearance of the R2 tyrosyl free radical. In parallel, there was an accumulation of apo-R2 protein in the inhibited cells. In vitro studies using pure, 59Fe-labeled recombinant mouse R2 protein unexpectedly showed that its iron center is labile at physiological temperatures and that iron is spontaneously lost from the protein even in the absence of chelators in a temperature-dependent process. Our conclusion is that parabactin or desferrioxamine inhibits ribonucleotide reduction and cell growth not by directly attacking the iron-radical center of the R2 protein, but instead by chelating the intracellular iron pool. This prevents the regeneration of the iron-radical center both in newly synthesized apo-R2 protein and in apo-R2 protein continuously formed from active R2 protein by the loss of iron.