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The blood-retinal barrier (BRB) and blood-brain barrier (BBB) protect, respectively, retina and brain from substances circulating in the blood that may affect the homeostatic regulation of the delicate and intricate neural microenvironment. In this project using both in-vivo and in-vitro model, I investigated the molecular properties of early stages of retina vascular development. Postnatal development of the murine vasculature in the retina affords the opportunity to explore mechanisms of BRB formation through use of tracers and pharmacological manipulation. To examine the onset of barrier function during post-natal vascular development, we modified a previously described in-vivo tracer assay (Poor et al, ARVO 2006). Fluorescent lysyl-dextrans were injected in the intraperitoneal cavity at different stages of development led to consistent filling of the retinal vasculature with tracer. 3kD fluorescent dextran freely extravasated from the retinal vasculature at post-natal day 5 (P5), but levels decreased at P8 and a complete barrier to the tracer was present by P10. Similar results were obtained with a larger tracer, 70 kD dextran, and a small 341u Dalton cross-linkable biotin (NHS-biotin). During the P9-P10 transition period, endothelial cell adhesion proteins such as Claudin-5 showed a distinct accumulation at cell-cell junctions. Pharmacological blockade of VEGF-A signalling, which is active at the growing vascular front, hastened barrier formation, suggesting that the BRB can only form following completion of the superficial vascular plexus and the concomitant down-regulation of VEGF-A levels. Phosphoproteomic analyses of the retina at early - P5 and later - P15 stage of development was performed in order to determine a key players in retina development. Acquisition of BRB function was also accompanied by a decrease in endothelial phospho-PAK and phospho-MLC, which is consistent with the decreased VEGF-A signalling. Use of an MLCK inhibitor also hastened barrier function, suggesting a critical need to regulate the acto-myosin cytoskeleton during formation of the BRB. Our findings provide a framework for the continued investigation of the inter- and intra-cellular changes required for BRB formation, and will hopefully yield insight into developing therapies to repair the barrier during neurovascular retinal.