Desmocollins (Dscs) and desmogleins (Dsgs) are cell-adhesion molecules involved in the formation of desmosome cell—cell junctions and share structural similarities to classical cadherins such as E-cadherin. In order to identify and provide quantitative information on the types of protein—protein interactions displayed by the type 2 isoforms and investigate the role of Ca²⁺ in this process, we have developed an Escherichia coli expression system to generate recombinant proteins containing the first two extracellular domains, namely Dsg2(1-2) and Dsc2(1-2). Analytical ultracentrifugation, chemical cross-linking, CD, fluorescence and BIAcore have been used to provide the first direct evidence of Ca²⁺ binding to desmosomal cadherins. These studies suggest that Dsc2(1-2) not only exhibits homophilic interactions in solution, but can also form heterophilic interactions with Dsg2(1-2). The latter, on the other hand, shows much weaker homophilic association. Our results further demonstrate that heterophilic interactions are Ca²⁺-dependent, whereas the Ca²⁺-dependence of homophilic association is less clear. Our data indicate that the functional properties of Dsc2(1-2) are more similar to those of classical cadherins, consistent with the observation that Dsc shares a higher level of sequence homology with classical cadherins than does Dsg. In addition to corroborating the conclusions of previously reported transfection studies which suggest the formation of lateral heterodimers and homodimers, our results also provide direct quantitative information on the strength of these interactions which are essential for understanding the adhesion mechanism.