The detachment of epithelial cells from the basal matrix during wound healing and differentiation of keratinocytes requires the disassembly of the hemidesmosomal multiprotein adhesion complex. Integrin alpha6beta4-plectin interaction plays a major role in the formation of hemidesmosomes, and thus the mechanisms regulating this interaction should be critical also for the disassembly process. Here we show that a particular plectin isoform (1a) interacts with the Ca(2+)-sensing protein calmodulin in a Ca(2+)-dependent manner. As a result of this interaction, binding of the hemidesmosome-associated plectin isoform 1a to integrin beta4 is substantially diminished. Calmodulin-binding inhibits also the interaction of plectin with F-actin. Further, we found that, during Ca(2+)-induced keratinocyte differentiation, plectin 1a is first relocated within the cell and later down-regulated, suggesting that Ca(2+) affects the fate of plectin 1a upon its release from hemidesmosomes. We propose a novel model for the disassembly of hemidesmosomes during keratinocyte differentiation, where both, binding of calmodulin to plectin 1a and phosphorylation of integrin beta4 by protein kinases, are required for disruption of the integrin alpha6beta4-plectin complex.