Insulin-degrading enzyme (IDE), which proteolytically degraded insulin with a high degree of specificity, was purified from pig skeletal muscle by ammonium sulfate precipitation, chromatography on Bio-Gel P-200 and DEAE-cellulose, and finally rechromatography on Sephadex G-200 (rechromatography fraction). The enzyme was also purified by affinity chromatography (affinity fraction). Both fractions migrated as a single component at the same position on polyacrylamidegel disc electrophoresis. Antiserum against pig muscle IDE was obtained by immunization of rabbits using the rechromatography fraction. By means of antiserum, it was shown that pig muscle IDE (affinity fraction), rat muscle cytosol-, and membrane-IDE gave a precipitin band of identity in Ouchterlony double-immunodiffusion systems. Quantitative immunoprecipitin data demonstrated that the antiserum inhibited the activities of the above three IDEs compared with normal rabbit serum. These data suggest that the insulin-degrading enzyme from porcine muscle and that from rat muscle have similar immunologic properties. The antiserum described here should be a useful tool for the examination of subcellular distribution and the quantitative analysis of insulin-degrading enzyme. It may also be helpful in determining the physiologic significance of IDE.