Raised vascular permeability precedes the clinical appearance of diabetic retinopathy and is closely correlated with the degree of hyperglycaemia and formation and accumulation of advanced glycation end products (AGEs) in the retinal vasculature. We have previously shown, using the isolated perfused retina, that retinal capillary permeability increased rapidly (< 30 s) in response to abluminal application of AGE-BSA via activation of the receptor for AGE (RAGE) and production of reactive oxygen species (ROS) from NADPH oxidase (Warboys & Fraser, 2005). We now report on the interaction between increased concentrations of glucose and AGE-BSA and the effects on retinal microvascular permeability. Retinae, obtained from Wistar rats, were mounted under a fluorescence microscope and continuously superfused with a Hepes buffer, pH 7.4, 37°C. The microvasculature was perfused with sulforhodamine dye via a micropipette inserted into a radial artery and permeability determined from the rate of decrease in fluorescence gradient across a venular capillary at zero flow and ambient pressure. A 15 min abluminal application of 25 mM glucose resulted in an initial (< 30 s) increase in permeability from 0.07 ± 0.03 to 1.27 ± 0.3 × 10^-6 cm.s^-1 (P < 0.01, paired t test, mean ± sem) that rapidly returned to basal values. This response was unaffected by RAGE blockade with 50 µg.ml^-1 anti-RAGE IgG (n = 4) or inhibition of NADPH oxidase with 100 µM apocynin (n = 10). No further increase in permeability was observed throughout the 15 min application, however, the response to AGE-BSA was significantly attenuated from 1.02 ± 0.1 to 0.31 ± 0.1 × 10^-6 cm.s^-1 (P < 0.001, n = 6). Similar treatments with osmotic controls were without effect. Furthermore, the response to AGE-BSA was also suppressed in ZDF rats, a model of Type 2 diabetes (blood glucose 21 ± 1.8 mM), compared to non-diabetic controls (blood glucose 5 ± 0.2 mM), with the maximal permeability response reduced from 1.34 ± 0.2 to 0.77 ± 0.4 × 10^-6 cm.s^-1 (P < 0.05, 2-way ANOVA, n = 3). The acute response to bradykinin (Bk) which requires ROS production, independent of NADPH oxidase activation, was unaffected by pre-treatment with 25 mM glucose. On the other hand, a 10 minute pre-treatment with AGE-BSA resulted in a leftward shift and elevation of the Bk dose-response curve suggesting potentiation of the acute response to Bk. This potentiation was abolished following inhibition of NADPH oxidase with apocynin. These data suggest a complex interplay between signal transduction pathways that are dependent on the rapid formation of ROS.