Histochemical demonstration of cathepsin B activity was performed for the soleus, extensor digitorum longus, cardiac and vascular smooth muscle tissues of the rat using CBZ-Arg-Arg-4-methoxy-beta-naphthylamide or CBZ-Ala-Arg-Arg-4-methoxy-beta-naphthylamide as the substrate. The enzyme varied in its apparent activity but was localized in discrete granules in all muscle types. Cathepsin B was most active in cardiac muscle and least active in extensor digitorum longus muscles in between these extremes similar to another lysosomal protease, dipeptidyl peptidase II. However, in both types of skeletal muscle, the granules were observed more frequently at the periphery of the muscle cell just beneath the sarcolemma. Since cathepsin B is found only in lysosomes, this subsarcolemmal predominence may indicate that only one population of lysosomes in muscle contains active cathepsin B. All cathepsin B activity was abolished in the presence of the protease inhibitor, leupeptin.