Disruption of epidermal-mesenchymal communication due to a delay in epithelialization, increases the frequency of developing fibrotic conditions in skin. As matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) are two key enzymes involved in wound healing and tissue remodeling, here we examined the efficacy of keratinocyte-fibroblast interaction on modulation of these enzymes and their inhibitors. The conditioned media derived from keratinocytes and fibroblasts grown in upper and lower chambers of a co-culture system, respectively, were analyzed for MMP-2 and -9. Keratinocyte or fibroblast conditioned medium (FCM) was used as a control. Gelatinolytic activity analyzed by zymography showed that keratinocytes mainly express MMP-9 and to a lesser extent MMP-2; while fibroblasts express only MMP-2. In a co-culture system, the activities of both MMP-2 and MMP-9 markedly increased in conditioned media collected from bottom chambers. These findings were consistent with the level of MMP-2 and MMP-9 measured by Western blot. Using the same experimental setting, the levels of tissue inhibitors of MMPs (TIMPs) secreted by keratinocytes and fibroblasts grown in the same co-culture system were also evaluated. Western blot showed that fibroblasts secrete only TIMP-1 and TIMP-2 whose levels were increased by co-culturing fibroblasts with keratinocytes. In contrary the level of TIMP-3, which was mainly expressed by keratinocytes, increased by co-culturing these cells with fibroblasts. In conclusion, interaction of fibroblast-keratinocyte modulates the levels of MMP-2 and -9 and their inhibitors produced by these cells and this interaction may be critical for a better healing quality at a late stage of the wound healing process. (Mol Cell Biochem 269: 209–216, 2005)