Histochemical localization of proteolytic activities in the dorsal epidermis of Japanese eel was demonstrated by fluorescent microscopy utilizing 4-methoxy-2-naphthylamide (4MβNA) derivatives as substrates and 5-nitrosalicylaldehyde as a trapping agent. Carbobenzoxy-L-phenylalanyl-L-arginyl-4MβNA (Cbz-Phe-Arg-4MβNA) and Cbz-Arg-Arg-4MβNA were used for direct detection of cathepsins L and B activities, respectively, in fresh frozen sections and unfixed cells of the eel epidermis. The fluorescing areas, where Cbz-Phe-Arg-4MβNA was hydrolyzed by cathepsin L, were shown in mucus secretory cells and club cells and broadly around skin surface. The fluorescing areas due to Cbz-Arg-Arg-4MβNA hydrolysis by cathepsin B were localized similarly in these tissues. The fluorescing intensity for both catheptic activities in mucus secretory cells was higher than that in club cells, where small fluorescing granules were distributed. These results indicate that eel cathepsins L and B are stored in epidermal secretory cells at different levels and probably serve as defense factors before or after secretion by these cells. Abbreviations: Cbz – carbobenzoxy; 4MβNA – 4-methoxy-2-naphthylamide; NSA – 5-nitrosalicylaldehyde.