The differentiation of keratinocytes involves numerous steps including the formation of the cornified envelope and the aggregation of keratin filaments by filaggrin monomer molecules. In this study, we investigated whether μ-calpain is involved in the processing of profilaggrin to filaggrin monomers by using both an active μ-calpain specific antibody and a 27-mer synthetic calpastatin peptide, a cell-permeable calpain-specific inhibitor. Upon incubation of cultured keratinocytes with Ca²⁺for 96 hours, active μ-calpain with a molecular mass of 76 kDa appeared preceded by an increase in μ-calpain mRNA. In synchrony with the appearance of active μ-calpain, the processing of profilaggrin occurred. Furthermore, the Ca²⁺-induced activation of μ-calpain and the processing of profilaggrin were affected by the addition of the synthetic calpastatin inhibitor. These results indicate that the activation of μ-calpain plays a major role in the profilaggrin processing.