The migration of lymphocytes from the bloodstream into lymph nodes (LNs) via high endothelial venules (HEVs) is a prerequisite for the detection of processed antigen on mature dendritic cells and the initiation of immune responses. The capture and arrest of lymphocytes from flowing blood is mediated by the multistep adhesion cascade, but the mechanisms that lymphocytes use to penetrate the endothelial lining and the basement membrane of HEVs are poorly understood. Matrix metalloproteinases (MMPs) control the metastatic spread of tumor cells by regulating the penetration blood vessel basement membranes. In this study, synthetic and natural inhibitors were used to determine the role of MMPs and MMP-related enzymes in regulating lymphocyte extravasation in mice. Mice were treated systemically with the hydroxamate-based MMP inhibitor Ro 31-9790 and plasma monitored for effective levels of Ro 31-9790, which block shedding of L-selectin. The total numbers of lymphocytes recruited into LNs were not altered, but L-selectin levels were higher in mice treated with Ro 31-9790. A reduced number of lymphocytes completed diapedesis and there was an increase in the number of lymphocytes in the endothelial cell lining, rather than the lumen or the basement membrane of HEVs. Lymphocyte migration and L-selectin expression in the spleen were not altered by Ro 31-9790 treatment. Two MMP inhibitors, TIMP1 and Ro 32-1541, did not block L-selectin shedding and had no effect on lymphocyte migration across HEVs. These results suggest that metalloproteinase activity is required for lymphocyte transmigration across HEVs into LNs and provide evidence for the concept that metalloproteinases are important players in some forms of transendothelial migration. (Blood. 2001;98:688-695)