Culture of human dermal fibroblasts within a three-dimensional matrix composed of native type I collagen fibrils is widely used to study the cellular responses to the extracellular matrix. Upon contact with native type I collagen fibrils human skin fibroblasts activate latent 72-kDa type IV collagenase/ gelatinase (MMP-2) to its active 59- and 62-kDa forms. This activation did not occur when cells were cultured on plastic dishes coated with monomeric type I collagen or its denatured form, gelatin. Activation could be inhibited by antibodies against MT1-MMP, by the addition of TIMP-2 and by prevention of MT1-MMP processing. MT1-MMP protein was detected at low levels as active protein in fibroblasts cultured as monolayers. In collagen gel cultures, an increase of the active, 60-kDa MT1-MMP and an additional 63-kDa protein corresponding to inactive MT1-MMP was detected. Incubation of medium containing latent MMP-2 with cell membranes isolated from fibroblasts grown in collagen gels caused activation of the enzyme. Furthermore, regulation of MT1-MMP expression in collagen cultures seems to be mediated by alpha2beta1 integrins. These studies suggest that activation of the proMMP-2 is regulated at the cell surface by a mechanism which is sensitive to cell culture in contact with physiologically relevant matrices and which depends on the ratio of proenzyme and the specific inhibitor TIMP-2.