The anion-exchange protein (band 3) reaction site in human erythrocytes for the fluorescent/phosphorescent probe eosinyl-5-maleimide (EMA) has been identified. Proteolytic dissection of band 3 in situ indicated that EMA reacts with the membrane-spanning Mr 17K peptide produced by chymotrypsin cleavage of band 3 in intact erythrocytes followed by removal of the cytoplasmic domain by mild trypsin digestion of ghost membranes. Sequencing of the major eosin-labeled peptide obtained from HPLC purification of an extensive chymotrypsin digest of purified Mr 17K peptide allowed assignment of the covalent reaction site for EMA to lysine-430 of the human erythrocyte protein [Tanner et al. (1988) Biochem. J. 256, 703-712]. Hydropathy plots based upon the primary structure of the protein [Lux et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9089-9093] suggest that this residue is in an extracellularly accessible loop connecting membrane-spanning segments 1 and 2 of native band 3 in the erythrocyte membrane. Inhibition of sequential labeling of intact erythrocytes by pairs of chemical probes including EMA, the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2-DIDS), and the reactively bifunctional spin-label bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA) has also been investigated. Each of these reagents affinity labels band 3 when added separately to a suspension of intact human erythrocytes by formation of one or more stable covalent bonds. Prelabeling of intact erythrocytes with EMA reduced subsequent labeling of band 3 by H2-DIDS by approximately 95% and by BSSDA by 90%. Similarly, prelabeling with H2-DIDS reduced subsequent labeling of band 3 by EMA by over 90%, and BSSDA prelabeling reduced EMA labeling by approximately 95%. Therefore, though having widely divergent chemical structures and protein modification reactivities, each of these negatively charged reagents may be competing for reaction with spatially overlapping sites on band 3 which are accessible from the extracellular space.