The molecular mass of clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, has been determined by analytical ultracentrifugation and gel exclusion chromatography. The result is in agreement with the formula mass of 16.8 kDa, demonstrating that the inhibitor is a monomer in aqueous solution. This enables the kinetics of unfolding and refolding to be interpreted in terms of folding in a kinetically two state, highly cooperative transition from the thermally unfolded state. (C) 2004 Published by Elsevier Inc.