Various low-molecular weight inhibitors of thiol proteinases have been isolated from actinomycetes and a mold, and Thiolstatin was recently reported from Bacillus cereus EY-21. Further screening for thiol proteinase specific inhibitors in microorganisms revealed inhibitory activity in the culture filtrate of strain 28Yl, which was identified as Nigrosabulum novosp. and produced a novel inhibitor, caricastatin, that specifically inhibits various thiol proteinases. Caricastatin was purified using Diaion HP-20, Amberlite IRC-50, and preparative HPLC. Physicochemical analyses (TLC, solubility, chemical reactions), hydrolysis experiments, mass spectrometry (FAB: M + H = 314), and 1H-NMR spectroscopy showed its structure to be Nα-acetyl-L-leucyl-D,L-arginal, analogous to thiolstatin D (with phenylalanine replaced by leucine). Inhibitory spectrum analysis demonstrated caricastatin is a potent inhibitor of thiol proteinases (papain, ficin, bromelain) and a very weak inhibitor of trypsin/trypsin-like serine proteinases, but does not inhibit other serine proteinases, metallo proteinases, carboxyl proteinases, peptidases, or non-proteolytic thiol enzymes. Though a novel microbial product, an identical compound was previously synthesized as a leupeptin analog.