Ribonucleotide reductase catalyzes a crucial step in de novo DNA synthesis and is allosterically controlled by relative levels of dNTPs to maintain a balanced pool of deoxynucleoside triphosphates in the cell. In eukaryotes, the enzyme comprises a heterooligomer of α₂ and β₂ subunits. The α subunit, Rnr1, contains catalytic and regulatory sites. Here, we report the only x-ray structures of the eukaryotic α subunit of ribonucleotide reductase from Saccharomyces cerevisiae. The structures of the apo-, AMPPNP only-, AMPPNP–CDP-, AMPPNP–UDP-, dGTP–ADP- and TTP–GDP-bound complexes give insight into substrate and effector binding and specificity cross-talk. These are Class I structures with the only fully ordered catalytic sites, including loop 2, a stretch of polypeptide that spans specificity and catalytic sites, conferring specificity. Binding of specificity effector rearranges loop 2; in our structures, this rearrangement moves P294, a residue unique to eukaryotes, out of the catalytic site, accommodating substrate binding. Substrate binding further rearranges loop 2. Cross-talk, by which effector binding regulates substrate preference, occurs largely through R293 and Q288 of loop 2, which are analogous to residues in Thermotoga maritima that mediate cross-talk. However loop-2 conformations and residue–substrate interactions differ substantially between yeast and T. maritima. In most effector–substrate complexes, water molecules help mediate substrate–loop 2 interactions. Finally, the substrate ribose binds with its 3′ hydroxyl closer than its 2′ hydroxyl to C218 of the catalytic redox pair. We also see a conserved water molecule at the catalytic site in all our structures, near the ribose 2′ hydroxyl.