Matrix metalloproteinases (MMPs) are hypothesized to play a key role in the processes of endothelial cell migration and matrix remodeling during angiogenesis. We utilized an in vitro model of microvascular endothelial cell angiogenesis, cells cultured within a collagen matrix, to investigate the MMP profile of endothelial cells undergoing angiogenesis. We demonstrated by gelatin zymography that monolayer cultures (two-dimensional) of endothelial cells constitutively expressed low levels of latent MMP-2, but that culture in a three-dimensional collagen matrix increased the total amount of MMP-2 mRNA and protein. Furthermore, 51% of total MMP-2 protein was activated in the three-dimensional culture lysates, compared with 3.5% in two-dimensional culture. The mRNA and protein of MT1-MMP, the putative activator of MMP-2, were up-regulated in endothelial cells cultured in three-dimensional as compared with two-dimensional culture. Treatment of cultures with MMP inhibitors blocked activation of MMP-2 and inhibited formation of endothelial cell networks within the collagen gel. Induction of MT1-MMP and MMP-2 appeared to be specific to collagen, inasmuch as culture of the endothelial cells on top of, or within, a Matrigel(R) matrix neither increased total MMP-2 nor increased activation of MMP-2. These results suggest that MT1-MMP activation of MMP-2 occurs in endothelial cells undergoing angiogenesis, that this activation has a functional role in endothelial cell organization, and that specific matrix interactions may be critical for the increased expression of MT1-MMP and MMP-2.