Triglyceride-rich lipoprotein (TGRL) lipolysis may provide a pro-inflammatory stimulus to endothelium. Detergent-resistant plasma membrane microdomains (lipid rafts) have a number of functions in endothelial cell inflammation. The mechanisms of TGRL lipolysis -induced endothelial cell injury were investigated by examining endothelial cell lipid rafts and production of reactive oxygen species (ROS). Lipid raft microdomains in human aortic endothelial cells were visualized using confocal microscopy with fluoroscein isothiocyanide-labeled cholera toxin B as a lipid raft marker. Incubation of Atto565-labeled TGRL with lipid raft-labeled endothelial cells showed that TGRL co-localized with the lipid rafts and TGRL lipolysis caused clustering and aggregation of lipid rafts and co-localization of TGRL remnant particles on the endothelial cells aggregated lipid rafts. Further, TGRL lipolysis caused translocation of low-density lipoprotein receptor-related protein, endothelial nitric oxide synthase, and caveolin-1 from raft regions to non-raft regions of the membrane 3 hours after treatment with TGRL lipolysis. TGRL lipolysis significantly increased the production of reactive oxygen species (ROS) in endothelial cells and both NADPH oxidase and cytochrome P450 inhibitors reduced production of ROS. Our studies suggest that alteration of lipid raft morphology and composition and ROS production could contribute to TGRL lipolysis-mediated endothelial cell injury.