Ribonucleoside diphosphate reductase from Escherichia coli consists of a 1/1 complex of two nonidentical subunits called proteins B1 and B2. The enzyme reduces the four common ribonucleoside diphosphates to the corresponding deoxyribonucleotides and is allosterically regulated by nucleoside triphosphates which influence its substrate specificity as well as its overall activity. The B1 subunit contains binding sites for the effectors while B2 contains iron and an organic free radical essential for catalytic activity. We now establish that only protein B1 binds substrates. Competition experiments support the presence of two identical substrate binding sites, distinct from the effector binding sites. The catalytic site of the enzyme thus is formed from both the B1 and B2 subunits. Dissociation constants for substrates ranged from 2 X 10(-5) to about 10(-3) M. In all cases effectors decreased these constants in agreement with their influence on the substrate specificity of ribonucleotide reductase, but did not induce cooperative effects. The increase in binding was pronounced at 20 degrees but only marginal at 0 degrees. Arrhenius plots of the influence of temperature on the catalytic activity of the enzyme showed sharp breaks at 12 degrees. The temperature effects can be interpreted as a conformational change occurring in the structure of protein B1 at the critical temperature.