The most common site (80%) of ovarian cancer metastasis is the omentum, a large (15 x 10 x 2 cm) peritoneal fold covering the small bowel. Because of the absence of model systems that accurately reproduce the microenvironment of the human omentum, the biological mechanism of early ovarian cancer metastasis is poorly understood. Using a new organotypic 3D culture of the omentum, we show that when cancer cells adhere, matrix-metalloproteinase (MMP)-2 is upregulated and proteolytically activated in these cells. The activated MMP-2 cleaves the matrix proteins fibronectin, vitronectin and collagen I into smaller fragments. The cleaved extra-cellular matrix (ECM) fragments then facilitate and accelerate cancer cell adhesion and invasion by binding to their cognate integrin receptors. In vivo inhibition of MMP-2 before adhesion by using a siRNA or a blocking antibody significantly reduced the number of metastasis and tumor weight in a xenograft mouse model. After metastasis had been established, blocking MMP-2 produced less of an effect. Our data identify tumor-derived proteolytically active MMP-2 as an early regulator of ovarian cancer metastasis.