Plasminogen activator inhibitor-1 is secreted bidirectionally by endothelial cells, acts as the primary regulator of fibrinolysis and as a key modulator of extracellular matrix proteolysis. Elevated serum levels of plasminogen activator inhibitor-1 are observed in serum of diabetic individuals. We investigated whether plasminogen activator inhibitor-1 is overexpressed in capillaries of diabetic donors with non-proliferative retinopathy compared to non-diabetic donors. We also assessed plasminogen activator inhibitor-1 expression in an animal model of retinopathy induced by exposing rabbit retinas to insulin-like growth factor-I. Colloidal gold immunocytochemistry was used to quantify plasminogen activator-1 antigen in donor retinas from diabetic subjects (n = 10) and control subjects (n = 10). This technique was also used to examine expression of plasminogen activator inhibitor-1 for correlation with retinal changes in the insulin-like growth factor-I-induced retinopathy model (n = 14). Plasminogen activator inhibitor-1 immunoreactivity was significantly increased in the retinas of all diabetic subjects as compared to controls. In the rabbit model, the expression of plasminogen activator inhibitor-1 immunoreactivity correlated with pathological retinal changes. In both the diabetic human and insulin-like growth factor-I-injected rabbit, overproduction of plasminogen activator inhibitor-1 was seen within the lumen of capillaries, within the cytoplasm of endothelial cells and in the basement membrane and extracellular matrix surrounding these capillaries. Minimal plasminogen activator inhibitor-1 was detected in the retinas of non-diabetics and in control rabbits injected with either heat-inactivated insulin-like growth factor-I or balanced salt solution. These studies support the conclusion that plasminogen activator inhibitor-1 is overexpressed in the retinal capillaries of diabetics with non-proliferative diabetic retinopathy and in rabbits with insulin-like growth factor-I-induced retinopathy.