alphaB-Crystallin is one of the six known mammalian small heat-shock proteins (sHsps). These are characterized by the presence of a conserved sequence of 80-100 residues, which constitutes the putative C-terminal domain. Like other sHsps, alphaB-crystallin forms multimeric globular complexes, often in combination with related sHsps. Here we show that in a yeast two-hybrid system, alphaB-crystallin can specifically interact with itself as well as with alphaA-crystallin and Hsp27. Analyses of the separate domains show that the conserved C-terminal domain (CalphaB) is essential for this interaction between subunits. To try and detect residues that are important in subunit interaction, the CalphaB domain was used in a two-hybrid screen as bait to select randomly mutated CalphaB mutants. In this way we obtained nine mutants that were still able to interact with wild-type CalphaB despite the presence of up to 15 replacements. Similarly, we obtained 16 mutants that were unable to bind, because of the presence of just three to nine replacements. In binding CalphaB mutants, lysine residues were most often replaced by glutamic acid residues, and in non-binding CalphaB mutants, acidic residues were often found to be replaced by non-charged residues. This indicates that negative charges are important for subunit interaction and we propose a model to explain this role of acidic residues. Furthermore, we observed that two homologs of alphaB-crystallin, alphaA-crystallin and Hsp27, generally interact similarly with the binding and non-binding CalphaB mutants as does alphaB-crystallin. This suggests that interactions involved in the complex formation of these three sHsps are largely comparable.