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Calpain is a ubiquitous neutral calcium-activated thiol protease that is implicated in various cellular functions including exocytosis, cell fusion, apoptosis and proliferation. The calpain system is composed of the enzymes mu-calpain and m-calpain and their endogenous inhibitor, calpastatin. We employed the spontaneously immortalized human HaCaT keratinocytes, which retain their ability to differentiate in vitro and in vivo, to study the modulation of the calpain system during keratinocyte differentiation. The cellular levels of keratinocyte differentiation markers and of the components of the calpain system were monitored by immunoblotting. Three established differentiation stimuli: increase in cell density as a function of time in culture, elevation of extracellular calcium concentration and exposure to 1,25-dihydroxyvitamin D3 enhanced the expression of the three keratinocyte differentiation markers keratin 10, involucrin and transglutaminase. The differentiation of HaCaT cells was accompanied by elevation of the components of the calpain system, although the pattern of increase varied according to the specific differentiation stimulus. A higher increase in calpains as compared with the increase in calpastatin suggests an increase in net calpain activity during differentiation. Such an increase may play a part in the differentiation process itself and/or in the regulation of key events in differentiating keratinocyte metabolism.