A zymographic assay for calpains in nondenaturing casein-containing polyacrylamide gels was developed. Calpain samples were run into the polyacrylamide gels by electrophoresis using a Tris-glycine buffer containing 1 mM EGTA to stabilize calpains. Upon completion of the electrophoresis, the gels were washed and incubated in a calpain activation buffer containing 1-4 mM calcium and 10 mM dithiothreitol for 20-24 h. After staining of the casein gels with Coomassie blue G250, both mu-calpain and m-calpain showed up as clearing bands. The amount of calpain loaded was proportional to the brightness of the clearing band. m-calpain can be easily distinguished from mu-calpain due to its higher mobility in the gel. Irreversible inhibitor (e.g., E64c) or tight-binding calmidazolium-treated mu-calpain remained inactive in the casein zymogram, whereas reversible inhibitor (e.g., calpain inhibitor I) was released from the protease by migration and dilution, lifting its inhibition. Crude homogenate of cultured cells (erythrocytes, Molt-4 and cerebrocortical neurons) or tissue (rat brain) can be directly analyzed for the presence of calpain isoforms despite the presence of endogenous calpastatin. Using this technique, mu-calpain activity in Molt-4 cells was found to decrease progressively with A23187 treatment, as a reflection of autolytic inactivation.