We have synthesized and characterized bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA), a membrane-impermeant bifunctional spin-labeling reagent. BSSDA is a nine carbon backbone homologue of bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate [BSSDP; Beth et al. (1986) Biochemistry 25, 3824-3832]. Due to its longer backbone, BSSDA can span longer distances between reactive groups on a protein than can BSSDP. However, the purpose of the bifunctional design of these reagents is to provide a tight motional coupling of the spin labels to the surface of a target protein. To test whether the longer backbone of BSSDA results in a greater local flexibility and thereby undermines the effects of bidentate attachment, we have labeled with BSSDA anion-exchange channels of intact human erythrocytes at the same site as we have previously labeled them with BSSDP. Linear and saturation-transfer EPR spectra of BSSDA-labeled anion-exchange channels in intact cells closely approximate the corresponding spectra from BSSDP-labeled channels. Thus, the longer backbone of BSSDA relative to BSSDP does not give rise to significant local flexibility, even when BSSDA is bound to a site that can be spanned by the shorter reagent.