One of the early features of diabetic retinopathy is the alteration of the blood-retinal barrier (BRB), which may involve the breakdown of endothelial cell tight junctions. The aim of this study was to examine the expression of extracellular proteinases in an animal model of early diabetic retinopathy and to determine their role in the alteration of the BRB. Matrix metalloproteinase (MMP) expression was studied in the retinas of rats with 12 weeks of diabetes. The role of MMPs in regulating tight junction function was investigated in retinal endothelial and pigment epithelial cells by measuring transepithelial electrical resistance (TER). The retinas of diabetic animals demonstrated elevated levels of MMP-2, MMP-9 and MMP-14 messenger RNA. A significant increase in the production of MMP-9 was seen when cells were exposed to high glucose conditions. Both cell types treated with purified MMP-2 or MMP-9 were found to have alterations of tight junction function as shown by decreased TER. Western blot analysis of cell extracts treated with MMP-2 or MMP-9, revealed specific degradation of the tight junction protein, occludin. Results suggest that elevated expression of MMPs in the retina may facilitate an increase in vascular permeability by a mechanism involving proteolytic degradation of the tight junction protein occludin followed by disruption of the overall tight junction complex.