Proteases are crucial for cancer metastasis, but due to lack of assays, their role in intravasation has not yet been tested. We have developed a human Alu sequence PCR-based assay to quantitate intravasated cells in an in vivo model. We demonstrated that metalloproteinases (MMPs), and most likely MMP-9, are required for intravasation by showing that marimastat, an inhibitor of MMPs, reduced intravasation by more than 90%, and that only tumor cell lines expressing MMP-9 intravasated. Cells with low surface urokinase plasminogen activator (uPA) and uPA receptor (uPAR) were also incapable of intravasation, despite the presence of high levels of MMP-9. We concluded that breaching of the vascular wall is a rate-limiting step for intravasation, and consequently for metastasis, and that cooperation between uPA/uPAR and MMP-9 is required to complete this step.