Teleost fish have a unique physical barrier composed of mucus and skin which act as the first line of defence.It has been shown that lymphocytes are present in the epidermis of fish,and the cutaneous mucus of several fish species contains immunoglobulin(Ig).As fish mucosal composition was complicated and the content of Ig was low,it is difficult to obtain high purity mucus Ig.In this study,skin mucus Ig of Japanese flounder(Paralichthys olivaceus)was purified by the combination of saturated ammonium sulfate precipitation,Sephacryl S300 gel filtration chromatography and DEAE Sepharose chromatography,and partial characteristics of purified proteins were analyzed by SDS-PAGE and Western-blot.According to SDSPAGE,many other proteins were removed from mucus by precipitation of 30% and 50% saturated ammonium sulfate solution,and Ig that was crudely extracted could reach a high purity by further purification using Sephacryl S300.After being finally purified by DEAE Sepharose chromatography,skin mucus Ig has only two bands of 72 and 26 ku,initially presumed as the heavy chain and light chain of Japanese flounder mucus Ig.Western-blot analysis showed that the monoclonal antibody produced from serum Ig could recognize the heavy chain of mucus Ig(72 ku).