The class I ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides and are composed of two subunits: R1 and R2. R1 contains the site for nucleotide reduction and the sites that control substrate specificity and the rate of reduction. R2 houses the essential diferric-tyrosyl radical (Y(*)) cofactor. In Saccharomyces cerevisiae, two R1s, alpha(n) and , have been identified, while R2 is a heterodimer (betabeta'). beta' cannot bind iron and generate the Y(*); consequently, the maximum amount of Y(*) per betabeta' is 1. To determine the cofactor stoichiometry in vivo, a FLAG-tagged beta ((FLAG)beta) was constructed and integrated into the genome of Y300 (MHY343). This strain facilitated the rapid isolation of endogenous levels of (FLAG)betabeta' by immunoaffinity chromatography, which was found to have 0.45 +/- 0.08 Y(*)/(FLAG)betabeta' and a specific activity of 2.3 +/- 0.5 micromol min(-1) mg(-1). (FLAG)betabeta' isolated from MMS-treated MHY343 cells or cells containing a deletion of the transcriptional repressor gene CRT1 also gave a Y(*)/(FLAG)betabeta' ratio of 0.5. To determine the Y(*)/betabeta' ratio without R2 isolation, whole cell EPR and quantitative Western blots of beta were performed using different strains and growth conditions. The wild-type (wt) strains gave a Y(*)/betabeta' ratio of 0.83-0.89. The same strains either treated with MMS or containing a crt1Delta gave ratios between 0.49 and 0.72. Nucleotide reduction assays and quantitative Western blots from the same strains provided an independent measure and confirmation of the Y(*)/betabeta' ratios. Thus, under normal growth conditions, the cell assembles stoichiometric amounts of Y(*) and modulation of Y(*) concentration is not involved in the regulation of RNR activity.