Removal of specific extracellular matrix (ECM) components has been implicated in the initiation of salamander limb regeneration. Remodeling of the ECM at the distal stump is necessary for the release of cells that eventually contribute to the blastema. Several matrix metalloproteinases (MMPs) have been well characterized as important to various physiological and pathological processes, such as bone remodeling and tumor invasion. The goal of this study is to identify and characterize MMPs that modulate the ECM during appendage regeneration in the axolotl Ambystoma mexicanum. By analyzing axolotl tissue extracts using gelatin-substrate gels, we have identified a 90-kDa gelatinase/collagenase that is upregulated within hours after limb amputation. This gelatinase shows a dramatic elevation in activity during the dedifferentiation and blastema stages. Its activity declines by the palette stage and returns to its basal level by the digit stages. The increase in activity of the 90-kDa gelatinase in response to amputation is independent of the nerve supply and the wound epithelium but these factors affect its subsequent downregulation. In addition to the blastema, the 90-kDa gelatinase can be detected in the stump at least 4 mm proximal to the regenerate. Similar regulation of the 90-kDa gelatinolytic activity is observed during tail regeneration and flank would healing. We suggest that this 90-kDa gelatinase/collagenase may play a role in the initiation and rapid growth phase of axolotl regeneration and wound healing.