Diabetic retinopathy (DR) commences as a basement membrane disorder with a dramatic loss of the innate retinal vascular autoregulation. In this process, retinal pericytes, which regulate endothelial cell proliferation and survival, undergo morphometric changes consistent with apoptosis. The ability of retinal pericytes to survive is dependent on their interaction with extracellular matrix (ECM) proteins, which are susceptible to rapid degradation by matrix metalloproteinases (MMPs). Here, we examined the potential involvement of MMPs and a membrane-type MMP in retinal pericyte death in experimental diabetes and in cultured retinal pericytes. Our data showed that chemically induced diabetes of 6 months' duration significantly increased the expression and activity of both MMP-2 and its physiological activator MT1-MMP. TdT-mediated dUTP nick end labeling (TUNEL)-positive pericytes and endothelial cells were concomitantly detected within the retinal capillaries of diabetic animals. In situ zymography showed a weak MMP activity in control retinas but an intense perivascular MMP activity in retinas from diabetic animals. In vitro studies showed that hyperglycemia-induced retinal pericyte apoptosis in vitro was attenuated by a specific MMP inhibitor. Incubation of pericytes with purified MMP-2 significantly increased the number of apoptotic cells. Our data suggest that increased MMP-2 activity compromises retinal pericyte survival possibly through MMP-2 action on ECM proteins and/or direct association of MMP-2 with integrins, which promotes apoptosis/anoikis by loss of cell contact with an appropriate ECM.