We investigated the interaction of the cesium ion (Cs + ) with the anionic intracellular components of human red blood cells (RBCs); the components studied included 2,3-bisphosphoglycerate (BPG), ADP, ATP, inorganic phosphate (P i ), carbonmonoxy hemoglobin (COHb), and RBC membranes. We used spin-lattice (T 1 ) and spin-spin (T 2 ) 1 3 3 Cs NMR relaxation measurements to probe Cs + binding, and we found that Cs + bound more strongly to binding sites in BPG and in RBC membranes than in any other intracellular component in RBCs at physiologic concentrations. By using James-Noggle plots, we obtained Cs + binding constants per binding site in BPG (66 +/- 8 M - 1 ), ADP (19 +/- 1 M - 1 ), ATP (25 +/- 3 M - 1 ), and RBC membranes (55 +/- 2 M - 1 ) from the observed T 1 values. We also studied the effect of Cs + on the oxygen (O 2 ) affinity of purified Hb and of Hb in intact RBCs in the absence and in the presence of BPG. In the absence of BPG, the O 2 affinity of Hb decreased upon addition of Cs + . However, in the presence of BPG, the O 2 affinity of Hb increased upon addition of Cs + . The O 2 affinity of Cs + -loaded human RBCs was larger than that of Cs + -free cells at the same BPG level. 3 1 P NMR studies on the pH dependence of the interaction between BPG and Hb indicated that the presence of Cs + resulted in a smaller fraction of BPG available to bind to the cleft of deoxyHb. Our NMR and O 2 affinity data indicate that a strong binding site for Cs + in human RBCs is BPG. A partial mechanism for Cs + toxicity might arise from competition between Cs + and deoxyHb for BPG, thereby increasing oxygenation of Hb in RBCs, and thus decreasing the ability of RBCs to give up oxygen in tissues. The presence of Cs + at 12.5 mM in intact human RBCs containing BPG at normal concentrations did not, however, alter significantly the O 2 affinity of Hb, thus ruling out the possibility of Cs + -BPG interactions accounting for Cs + toxicity in this cell type.