An enzyme capable of degrading insulin was purified from pig skeletal muscle and studied for its characteristics. Purification of the enzyme was successfully achieved by a combination of ammonium sulfate precipitation, chromatography of Bio-Gel P-200 and DEAE-cellulose, and, finally, ampholine isoelectrofocusing. The enzyme obtained showed 741-fold purification in its activity and a single band on polyacrylamide gel electrophoresis. The purified enzyme degraded insulin proteolytically and was sulfhydryl dependent. The Km for insulin was 70 nM. Proinsulin behaved as a competitive inhibitor for insulin degradation; its Ki was 320 nM. Glucagon was also proteolytically degraded, whereas number of other peptides, including A and B chains of insulin, were not appreciably affected by this enzyme. The molecular weight of the enzyme was estimated to be 135,000 by gel filtration on Sephadex G-200 and 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme and lysosomal enzymes extracted from rat liver were shown to be different, judging from their optimal pH for insulin degradation and substrate specificity. These studies demonstrate the presence of an insulin-degrading enzyme in pig skeletal muscle and suggest that this enzyme is identical to rat insulin protease in most of its biochemical properties.