New thiol protease inhibitors, estatins A and B, were isolated from the culture filtrate of Myceliophthora thermophila M4323. The basic, water-soluble inhibitors were characterized as having an agmatine, /m^-epoxysuccinic acid and L-phenylalanine or L-tyrosine moieties in the structure. The molecular formulas C18H25N5O5and C18H25N5O6for A and B were indicated by elemental analysis and fast atom bombardment MS. Estatins were specific inhibitors against thiol proteases such as papain, ficin and bromelain. They suppressed IgE antibody production in mice, but not IgG.Lowmolecular weight protease inhibitors are useful at least as medicines or reagents for research.During our screening for thiol protease-specific inhibitors, a fungal culture produced inhibitors against papain. The inhibitors were isolated and named estatins A and B (Fig.