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Purpose. To investigate the effect of endothelial cells (EC) on the barrier function of the retinal pigment epithelium (RPE).Methods. Primary bovine RPE were grown in solo culture or in coculture with bovine EC. Culture media of RPE were varied to develop a monolayer with stable barrier properties determined by transepithelial electrical resistance (TER) and permeability to sodium fluorescein. The effect of EC on the barrier properties of RPE was tested in contacting and non-contacting cocultures of RPE and EC. The conditioned media of cocultures were analysed for soluble vascular endothelial growth factor (VEGF) by ELISA. A neutralizing antibody to VEGF165 was added to cocultures of RPE and EC and the TER was measured.Results. RPE had maximal barrier properties (high TER, low permeability, positive staining for barrier proteins) at day 10 that persisted until day 20. Compared to solo RPE culture, cocultivation of RPE with EC reduced RPE barrier function significantly and led to a greater release of soluble VEGF into the conditioned media (p<0·05). Neutralizing VEGF with antibody led to partial recovery of barrier properties in the coculture conditions (p<0·03).Conclusions. Coculture of RPE with EC reduces RPE barrier properties and the reduction is, in part, mediated by soluble VEGF. EC–RPE contact-induced disruption of barrier properties occurs in ocular pathologies such as choroidal neovascularization, where EC move through Bruch's membrane and contact the RPE, leading to further exacerbation of the already compromised blood-retinal barrier.