Uveitis is a systemic immune disease and a common cause of blindness. The eye is an ideal organ for light-based imaging of molecular events underlying vascular and immune diseases. The phospholipid, platelet activating factor (PAF), is an important mediator of inflammation, the action of which in endothelial and immune cells in vivo is not well understood. Using our recently introduced in vivo molecular imaging approach in combination with PAF-inhibitors WEB 2086 (WEB) and Ginkgolide B (GB), we show a key role for PAF in endothelial injury in uveitis. The differential inhibitory effects of WEB and GB in reducing LPS-induced endothelial injury in the choroid indicate an important role for PAF-like lipids, which might not require the PAF-receptor for their signaling. PSGL-1-mediated rolling of mouse leukocytes on immobilized Pselectin in our autoperfused microflow chamber assay revealed a significant reduction in rolling velocity upon the cells’ contact with PAF. Rolling cells that came in contact with PAF rapidly assumed morphological signs of cell activation, indicating that activation during rolling does not require integrins. Our results show a key role for PAF in mediating endothelial and leukocyte activation in acute ocular inflammation. Our in vivo molecular imaging provides a detailed view of cellular and molecular events in the complex physiological setting.