Background: Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have important functions during skin development, repair and maintenance. MMP-2 and MMP-9 (gelatinase A and gelatinase B) are involved in regulating keratinocyte migration. Objectives: To analyse whether ApligrafR, a bilayered tissue-engineered human skin equivalent (HSE), produces gelatinases and TIMPs and whether or not epidermal-dermal interactions regulate MMP activity. Methods: The tissue distribution of MMP-2, MMP-9, TIMP-1, TIMP-2 and (r)bronectin was analysed by immunohistochemistry. Secreted MMP activity was quantified by a fluorimetric assay and gelatin zymography was used to monitor gelatinases in tissue culture supernatants. Results: ApligrafR expressed MMP-2 and MMP-9 and contained immunohistochemically detectable amounts of TIMP-1 and TIMP-2. The gelatinases were predominantly produced in the epidermis, whereas immunostaining of TIMP-1 and TIMP-2 was largely confined to the dermal component of the HSE. Fibronectin was expressed only in the dermis. Gelatin zymography demonstrated that intact ApligrafR produced both MMP-2 and MMP-9, the latter predominantly in its latent form. Separation of the dermis from the epidermis resulted in an enhanced production and activation of MMP-9 by the epidermal layer, and secretion of latent and active MMP-2 by the dermal layer. Moreover, the incubation media of the separated epidermis demonstrated significantly stronger MMP activity than did intact ApligrafR or its dermal component. Conclusions: These observations provide evidence that epidermal-dermal interactions suppress epidermal gelatinase activity. In addition, coexpression of TIMPs and fibronectin in the ApligrafR dermis suggests that the product has the potential to counteract the imbalance between matrix production and degradation in chronic wounds and thus may support wound re-epithelialization.