The β(2) subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe(2)(III)-Tyr(•)) that is essential for nucleotide reduction. The β(2) subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β'). Although only β is capable of iron binding and Tyr(•) formation, cells lacking β' are either dead or exhibit extremely low Tyr(•) levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β' is required for iron loading and Tyr(•) formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr(•)-reducing agent hydroxyurea. Transient induction of β' in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr(•) levels in β. Tyr(•) can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β' and is inhibited by adding an iron chelator prior to, but not after, β' supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr(•) levels and ββ' activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr(•) cofactor in RNR.