A new family of cysteine protease inhibitors from the basidiomycete Macrolepiota procera has been identified and the family members have been termed macrocypins. These macrocypins are encoded by a family of genes that is divided into five groups with more than 90% within-group sequence identity and 75-86% between-group sequence identity. Several differences in the promoter and noncoding sequences suggest regulation of macrocypin expression at different levels. High yields of three different recombinant macrocypins were produced by bacterial expression. The sequence diversity was shown to affect the inhibitory activity of macrocypins, the heterologously expressed macrocypins belonging to different groups showing differences in their inhibitory profiles. Macrocypins are effective inhibitors of papain and cysteine cathepsin endopeptidases, and also inhibit cathepsins B and H, which exhibit both exopeptidase and endopeptidase activities. The cysteine protease legumain is inhibited by macrocypins with the exception of one representative that exhibits, instead, a weak inhibition of serine protease trypsin. Macrocypins exhibit similar basic biochemical characteristics, stability against high temperature and extremes of pH, and inhibitory profiles similar to those of clitocypin from Clitocybe nebularis, the sole representative of the I48 protease inhibitor family in the merops database. This suggests that they belong to the same merops family of cysteine protease inhibitors, the mycocypins, and substantiates the establishment of the I48 protease inhibitor family.