Collagenolytic activity has been reported previously in association with wounds. We used in situ hybridization and immunohistochemistry to localize cellular sites of interstitial collagenase production in acute wounds in human skin at days 1, 2, 4, 6, 9, and 14 after wounding. In vivo, collagenase expression peaked in migrating basal keratinocytes at the wound edge at day 1, then gradually decreased and was undetectable at day 9 when healing was complete. To minimize the effects of crust formation and inflammation, we examined the healing of wounds made with a 3-mm punch in organ-cultured skin. In these in vitro wounds, re-epithelialization occurred by 5-7 d in 10% serum, although remodeling of the connective tissue was minimal. Collagenase expression showed a similar pattern as in the in vivo wounds; it was detected in migrating keratinocytes already 4-6 h after wounding, peaked at 12-24 h, gradually decreased during the next few days, and subsided upon re-epithelialization. In dermal fibroblasts, on the other hand, expression of collagenase started considerably later, after 5-7 d in culture, and persisted after complete re-epithelialization, indicating that collagenase is differentially regulated in different cell types. Our findings also show that collagenase induction in keratinocytes does not require inflammation and occurs as a rapid response to wounding, suggesting that interstitial collagenase is not only necessary for remodeling of the extracellular matrix, but may also have a role in initiating migration of keratinocytes in wound healing.