Matrix metalloproteinases (MMPs) play a key role in vascular remodeling and cardiovascular disease. MMPs degrade and reorganize the vascular extracellular matrix (ECM). In atherosclerosis MMP activity is associated with plaque rupture leading to heart attacks and stroke. Surprisingly little is known about the targets of MMPs in the vasculature. In the present study, we used a proteomics approach to identify vascular targets for three members of the major classes of MMPs: MMP-3 - a member of matrilysins, MMP-9 - a gelatinase and MMP-14 - a member of the membrane–bound MMPs. We incubated human radial arteries with MMP-3, -9 and -14 and analyzed the released proteins by gel-LC-MS/MS. New substrates were confirmed by digestion of recombinant proteins with the same MMPs. Further analysis focused on the identification of new MMP cleavage sites of ECM proteins by searching for non-tryptic peptides. Finally, the newly identified degradation products were validated in human aortic tissue with high MMP-9 activity to relate protein degradation in vitro with exogenous MMPs to endogenous proteolytic activity in vivo. Using this innovative approach, we identified 17 novel targets, including ECM proteins associated with the basement membrane (collagens, nidogen-1, agrin), elastic fibers (emilin-1, transglutaminase-2, TGF β-induced protein ig-h 3) and other extracellular proteins (galectin-1, tenascin-X, tenascin-C). In total, we detected 74 cleavage sites for MMP targets, many of which were shared among different MMPs. Thus, our current classification of MMPs based on few selected targets (collagenases, elastase, etc) is an oversimplification of a complex area of biology, and our study is a first attempt to contribute to a better understanding about the role of MMPs in the vasculature.