We compared the in vitro degradation of porcine and human insulin in the subcutaneous tissue of rat. The insulin degrading activity was largely confined to the 160000×g supernatant fraction of subcutaneous tissue. The degradation of human insulin was approximately half that of porcine insulin in the supernatant fraction. The degradation of porcine insulin in subcutaneous tissue was inhibited by bacitracin, leupeptin, phosphoramidon, and Z-Gly-Pro-Leu-Gly, though the human insulin degradation was not. The degradation of both insulins was accelerated by glutathione. While the proteolytic enzyme activities of cathepsin-B and collagenase-like peptidase were detectable in subcutaneous tissue, chymotrypsin, elastase, kallikrein, α-thrombin, and trypsin activities were almost negligible. These in vitro studies suggest that human insulin is comparatively stable against proteolytic enzymes, probably collagenase-like peptidase or cathepsin-B, in the subcutaneous tissue, which support the in vivo evidence.