Proteolysis is an intrinsic component of cutaneous wound repair and several matrix metalloproteinases have been shown to participate in various stages of this process. Therefore, we investigated the expression of a novel metalloproteinase, collagenase-3 (MMP-13), in normally healing cutaneous wounds and chronic venous ulcers. MMP-13 was expressed abundantly by fibroblasts deep in the chronic ulcer bed but was not detected in epidermis and all the acute wounds. The spatial expression of MMP-13 differed from that of collagenase-1 (MMP-1), which was prominently expressed by migrating keratinocytes and dermal cells located just beneath the wound surface. Northern blot hybridization did not reveal expression of MMP-13 by fibroblasts cultured on tissue culture plastic. In accordance with our in vivo findings, however, fibroblasts grown in a collagen gel produced MMP-13 mRNA abundantly. Our results suggest that MMP-13 can be induced in skin during wound repair after altered cell-matrix interactions. Although both MMP-1 and MMP-13 have the unique ability to degrade fibrillar collagens, their regulation and role during wound repair seem different. Collagenase-1 is critical for re-epithelialization, and MMP-13 most likely plays a role in the remodeling of collagenous matrix in chronic wounds.