The hemoglobin regulator, 2,3-bisphosphoglycerate (glycerate-2,3-P2) has been shown to modulate the activity of casein kinase II from rabbit reticulocytes. Kinetic results were obtained with the exogenous substrate, beta-casein and the endogenous substrates, initiation factors (eIF-) 2 and 3. Experiments carried out to determine the interaction between glycerate-2,3-P2, Mg2+, substrate, and casein kinase II led to the following conclusions: 1) glycerate-2,3-P2 inhibition was competitive with respect to the protein substrate and noncompetitive with respect to ATP; 2) inhibition was not caused by depletion of ATP-Mg2+ as a consequence of Mg2+ complexation with glycerate-2,3-P2; 3) the response curve for glycerate-2,3-P2 was cooperative, but the cooperativity decreased as salt concentration increased; 4) glycerate-2,3-P2 inhibition was dependent on Mg2+ concentration up to about 5 mM MgCl2 but did not parallel glycerate-2,3-P2 X Mg2+ complex formation indicating that the Mg2+ dependence was not due to the formation of a glycerate-2,3-P2 X Mg2+ complex; 5) experiments with analogs of glycerate-2,3-P2 showed that the binary phosphate grouping was important in determining inhibition by glycerate-2,3-P2 while the presence of the carboxylate-phosphate pair was much less important; 6) low levels of glycerate-2,3-P2 stimulated phosphorylation of beta-casein, eIF-2, and eIF-3; the extent of stimulation was dependent on the affinity for casein kinase II and the level of the substrate. These effects were observed in the range of glycerate-2,3-P2 concentrations predicted for intracellular fluctuations in this metabolite. Therefore, it was concluded that glycerate-2,3-P2 could function both as an activator and an inhibitor of casein kinase II in the erythroid cell by binding at the substrate binding site.