Spectrin tetramers form by the interaction of two α–β dimers through two helices close to the C-terminus of a β subunit and a single helix at the N-terminus of an α subunit. Early work on spectrin from solid tissues (typified by αII and βII polypeptides) indicated that it forms a more stable tetramer than erythroid spectrin (αI–βI). In the present study, we have probed the molecular basis of this phenomenon. We have quantified the interactions of N-terminal regions of two human α polypeptides (αI and αII) with the C-terminal regions of three β isoforms (βIΣ1, βIIΣ1 and βIIΣ2). αII binds either βII form with a much higher affinity than αI binds βIΣ1 (Kd values of 5–9 nM and 840 nM respectively at 25 °C). βIIΣ1 and βIIΣ2 are splice variants with different C-terminal extensions outside the tetramerization site: these extensions affect the rate rather than the affinity of α subunit interaction. αII spectrin interacts with each β subunit with higher affinity than αI, and the βII polypeptides have higher affinities for both α chains than βIΣ1. The first full repeat of the α subunit has a major role in determining affinity. Enthalpy changes in the αII–βIIΣ2 interaction are large, but the entropy change is comparatively small. The interaction is substantially reduced, but not eliminated, by concentrated salt solutions. The high affinity and slow overall kinetics of association and dissociation of αII–βII spectrin may suit it well to a role in strengthening cell junctions and providing stable anchor points for transmembrane proteins at points specified by cell-adhesion molecules.