ABSTRACT Ribonucleotide reductase (RNR) is an enzyme that catalyzes the conversion of ribonucleotides to C2ʹ-deoxyribonucleotides, the building blocks for both DNA synthesis and repair in all living organisms, via controlled radical chemistry. Since RNR expression is elevated in many cancers, this enzyme is a major target of anticancer drugs. Until recently, the only clinically proven RNR-inhibition pathway was a suicide inactivation in which synthetic substrate analogues, for instance, gemcitabine diphosphate (F2CDP), irreversibly inactivate the RNR-α2β2 heterodimeric complex. Recently, Clofarabine (ClF) nucleotides have been shown to reversibly inhibit the enzyme by targeting the large subunit of RNR (RNR-α) and inducing RNR- α-persistent hexamerization. To date, ClF nucleotides are the only known examples of this mechanism. In order to establish whether this reversible inhibition is a common mode of inhibition among nucleotide-derivative drugs, the active forms of two other drugs, Cladribine (ClA) and Fludarabine (FlU), were chemoenzymatically synthesized and their mechanism(s) of inhibition of RNR were evaluated. Enzyme inhibition and fluorescence anisotropy assays show that di- and triphosphates of these two nucleosides reversibly inhibit RNR with diverse Ki values, fairly dispersed in a range of 0.5–10 μM, and bind to the catalytic site (C-site) and the allosteric activity site (A- site) of RNR-α, respectively. Our EM studies, gel filtration, FRET, and protease digestion assays suggest that RNR-inhibition is coupled with the formation of conformationally distinct hexamers. Studies in Flp-In HEK293 T-REx cells capable of selectively inducing either wild-type or oligomerization-defective mutant RNR-α overexpression define the central role of RNR-α oligomerization in drug activity, and highlight a potential resistant mechanism to these drugs. The results of these studies set the stage for new interventions targeting RNR oligomeric regulation.