The purpose of this study was to examine the appearance and activation of collagenase-1 (MMP-1) in the wound environment. We found that MMP-1 accumulates in the fluid phase of the burn wound environment within 2 d of injury and reaches maximal levels by day 4. Two forms of the enzyme were evident; one that corresponded to proMMP-1 and another that corresponded to a group of high molecular mass (approximately 200 kDa and >200 kDa doublet) MMP-1 containing complexes. ProMMP-1 and MMP-1 containing complexes also occurred in wound fluid from venous stasis ulcers, but neither was detected in mastectomy fluid or in plasma. Levels of the proteinase inhibitor alpha2-macroglobulin in burn fluid and chronic ulcer wound fluid were almost as high as in plasma, and the high molecular mass MMP-1 containing complexes in burn fluid appeared to result from binding between alpha2-macroglobulin and activated MMP-1. These observations provide direct evidence that active MMP-1 in the fluid phase of the wound environment becomes complexed to alpha2-macroglobulin.