Calpain is a Ca2 + -dependent cysteine protease which is found in the microsomal and cytosolic compartments of most mammalian neurons and other cells1}. Calpain hydrolyses peptide bonds of proteins in the cytosol2'3). A large amount of research effort has been focused on this enzyme, since it potentially plays a central role in several physiological events, such as turnover of myofibrillar proteins, protein kinase C activation, cytoskeleton and cell membrane organization2*, neuropeptide metabolism2), and activation of platelets3*. Inhibitors of calpain prevent the breakdown of cytoskeletal proteins induced by calcium efflux in intact cells4'5). Consequently they could potentially be used in the treatment of neurodegenerative and muscular dystrophies diseases. In the course of screening for potential calpain inhibitors from microbial extracts6), an actinomycete strain, (SC433), exhibited activity in a calpain-casein assay using 3H-casein as the substrate7). A new bioactive secondary metabolite named phevalin, was isolated from the ethyl acetate extract of this culture. This compound is a new pyrazinone calpain inhibitor derived from phenylalanine and valine. This report presents the production and structure determination of phevalin, mainly by detailed spectroscopic methods including MS, ID and 2D NMR.