The first committed step in DNA biosynthesis occurs by direct reduction of ribonucleotides. This reduction is catalyzed by ribonucleotide reductase (RNR), an enzyme which uses a unique radical mechanism to facilitate the transformation. All four DNA precursors are synthesized by a single enzyme. Therefore, an intricate pattern of regulation has evolved to insure that RNR generates the proper quantity of each deoxyribonucleotide. It is this regulation, and conditions that influence this regulation, that are the central focal points of this dissertation. The studies described in this thesis have been aided by the development of a novel RNR assay. Unlike the traditional assay, this new procedure permits the simultaneous monitoring of all four RNR activities. This four-substrate assay was used to investigate whether the four enzyme activities of RNR were differentially sensitive to inhibition by the radical scavenger, hydroxyurea. The assay results, along with the results of a technique that measured enzyme inhibition as a function of radical decay, suggest that all activities of RNR are equally inhibited by hydroxyurea. Instead of differential inhibition, it appears that the activity level of RNR determines the relative sensitivity to hydroxyurea. The effects of nucleotide effectors and substrates on the relative turnover rates of the vaccinia virus and T4 phage RNR were also investigated by use of the four-substrate assay. When physiological concentrations of the allosteric effectors and substrates were added to the reaction mixtures, both enzyme forms produced dNDPs in ratios that approximate the nucleotide composition of their respective genomes. Non-physiological nucleotide concentrations generated significantly different product profiles, indicating that RNR has evolved to function within a defined nucleotide environment. Interestingly, the substrate component of the nucleotide environment proved to be as important as the allosteric effectors in modulating the reaction rates. Although the allosteric effects of nucleoside triphosphates have been known for some time, little attention has been given to the potential role that substrates play in the regulation of RNR. The results from my research suggest that the regulation of RNR in vivo results from a complex interplay between the enzyme and its substrates, products, and allosteric effectors. / Graduation date: 1998