We have developed a new membrane-impermeant, bifunctional spin-labeling reagent, bis- (sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP), and employed it in an electron paramagnetic resonance (EPR) study of the rotational diffusion of the anion-exchange channel (band 3) in intact human erythrocytes. BSSDP reacts in a covalent manner and with high specificity with the extracytoplasmic domain of band 3, forming a complex in which the spin-label is immobilized on the protein. The linear EPR spectrum of BSSDP-labeled intact erythrocytes is characteristic of a highly immobilized, spatially isolated nitroxide probe. The saturation-transfer EPR spectrum of the same sample indicates that the anion channel in intact erythrocytes exhibits rotational dynamics in the 0.1-1 ms correlation time range at 20 °C. Rotational dynamics in this motional domain are consistent with a strong interaction of the anion-exchange channel with the erythrocyte cytoskeleton. The saturation-transfer EPR spectrum of ghosts prepared from BSSDP-labeled erythrocytes indicates a significant increase in rotational mobility of the anion channel, suggesting a significant disruption on lysis of interactions between the anion channel and the cytoskeleton.