Desmosomes in tissues are resistant to disruption by chelation of extracellular calcium. It has been suggested that this represents a hyper-adhesive state of these intercellular junctions that is crucial for the maintenance of epidermal integrity. Desmosomes change to a lower affinity, calcium-dependent adhesive state when cells are cultured at low density or when an intact epithelial cell sheet is wounded. Here we demonstrate that cells of the immortalized human keratinocyte line HaCaT acquire calcium-independent desmosomes in confluent culture. An adhesion assay shows that HaCaT cells with calcium-independent desmosomes are more cohesive than cells with calcium-dependent desmosomes. This assay relates directly to desmosomal adhesion because it involves splitting of the desmosomal adhesive material. Moreover, the difference in adhesiveness between calcium-dependent and calcium-independent desmosomes involves no quantitative change in the known protein composition of desmosomes. Instead, switching between the two adhesive states can be achieved by activation or inhibition of protein kinase C (PKC), suggesting a direct effect of PKC signalling on desmosomal adhesion. These results provide direct support for the concept of hyper-adhesiveness in desmosomes.