The spectrin heterodimer is formed by the antiparallel lateral association of an alpha and a beta subunit, each of which comprises largely a series of homologous triple-helical motifs. Initiation of dimer assembly involves strong binding between complementary motifs near the actin-binding end of the dimer. In this study, the mechanism of lateral spectrin association at this dimer nucleation site was investigated using the analytical ultracentrifuge to analyze heterodimers formed from recombinant peptides containing two or four homologous motifs from each subunit (alpha20-21/beta1-2; alpha18-21/beta1-4). Both the two-motif and four-motif dimer associations were weakened substantially with increasing salt concentration, indicating that electrostatic interactions are important for the dimer initiation process. Modeling of the electrostatic potential on the surface of the alpha20 and beta2 motifs showed that the side of the motifs comprising the A and B helices is the most favorable for association, with an area of positive electrostatic potential on the AB face of the beta2 motif opposite negative potential on the AB face of the alpha20 motif and vise versa. Protease protection analysis of the alpha20-21/beta1-2 dimer showed that multiple trypsin and proteinase K sites in the A helices of the beta2 and alpha21 motifs become buried upon dimer formation. Together, these data support a model where complementary long range electrostatic interactions on the AB faces of the triple-helical motifs in the dimer nucleation site initiate the correct pairing of motifs, i.e. alpha21-beta1 and alpha20-beta2. After initial docking of these complementary triple-helical motifs, this association is probably stabilized by subsequent formation of stronger hydrophobic interactions in a complex involving the A helices of both subunits and possibly most of the AB faces. The beta subunit A helix in particular appears to be buried in the dimer interface.