The allosteric effectors, ATP, dATP, dGTP and dTTP, regulate both the level of activity and the substrate specificity of ribonucleoside diphosphate reductase from Escherichia coli. The active enzyme consists of a complex of two non-identical subunits, proteins B1 and B2. Addition of negative effectors, such as dATP, leads to the formation of an inactive form which is probably a dimer of the active enzyme. We have now studied the binding of the effector nucleotides to the B1 and B2 subunits. Subunit B2 does not bind any of the nucleotides, while subunit B1, either alone or in combination with subunit B2, contains a total of four binding sites. These can be divided into two classes, each containing two sites. The first class (h-sites) has a high affinity for dATP and binds ATP, dATP, dGTP and dTTP; the second class (l-sites) has a lower affinity for dATP and binds only ATP and dATP. Binding of dATP to l-sites is considerably tighter in the presence of protein B2 than in the isolated protein B1. This suggests a mechanism for the generation of the inactive form of the enzyme through the binding of dATP. It appears that the l-sites are primarily involved in the regulation of the over-all activity of the enzyme, while binding of effectors to the h-sites influences the substrate-specificity. The binding of different nucleotides to h- and l-sites results in the formation of many distinct species of protein-effector complexes, each of which can be related to particular functional states of the enzyme. This suggests that the regulation of ribonucleoside diphosphate reductase requires the existence of many distinct conformational states.