Spectrin assembles into an anti-parallel heterodimeric long flexible rod-like molecule through a multi-step process initiated by a high affinity interaction between discrete complementary homologous motifs near the actin binding domain. To date, attempts to determine a 3-dimensional structure of this critical complex using crystallography have been unsuccessful; therefore, in this study we used homology modeling coupled with structural refinement based on distance constraints from chemical crosslinking to determine the subunit-subunit docking interface and a plausible medium resolution structure of the heterodimer initiation site. Intramolecular and intermolecular crosslinks formed by the “zero length” cross-linking reagent, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) were identified after trypsin digestion of the crosslinked heterodimer complex using LC-MS/MS analysis. High confidence assignment of crosslinked peptides was facilitated by determination of crosslinked peptide masses with an uncertainty of only a few parts per million using a high sensitivity linear ion trap mass spectrometer equipped with a Fourier-transform ion cyclotron resonance (FT-ICR) detector. Six interchain crosslinks were able to clearly distinguish among three likely docking models, and these distance constraints, as well as two intrachain crosslinks in the {alpha]-subunit and one intrachain crosslink in the -subunit, were used to further refine an initial homology based structure. The final model is consistent with all available physical data including protease protection experiments, isothermal titration calorimetry analyses, and location of a common polymorphism that destabilizes dimerization. This model supports the hypothesis that initial docking and pairing of the correct and motifs from among many very similar motifs in both subunits is driven primarily by long range electrostatic interactions.