AbstractThe ubiquitous calpains, µ‐ and m‐calpain, are implicated in a variety of vital (patho)physiological processes and therefore cell‐permeable specific inhibitors represent important tools for defining the role of calpains in cells and animal models. A syntheticN‐acetylated 27‐mer peptide derived from exon B of the human calpastatin inhibitory domain 1 is known to be the most potent and selective reversible inhibitor of calpains. To improve the membrane permeability of this peptidic inhibitor, it wasN‐terminally extended with or disulfide‐linked to theC‐terminal 7‐mer fragment of penetratin, a well‐established vector for cell membrane translocation of bioactive compounds. Despite the shorter penetratin sequence, both constructs showed increased cell permeability and retained their full calpain inhibitory potency. Copyright © 2006 European Peptide Society and