Atherosclerosis originates as focal arterial lesions having a predictable distribution to regions of bifurcations, branches and inner curvatures where blood flow characteristics are complex. Distinct endothelial phenotypes correlate with regional hemodynamics. We propose that systemic risk factors modify regional endothelial phenotype to influence focal susceptibility to atherosclerosis. Transcript profiles of freshly isolated endothelial cells from 3 athero-susceptible and 3 athero-protected arterial regions in adult swine were analyzed to determine the initial pre-lesional effects of hypercholesterolemia upon endothelial phenotypes in vivo. Cholesterol efflux transporter ABCA1 was upregulated at all sites in response to short-term high fat diet. Pro-inflammatory and anti-oxidative endothelial gene expression profiles were induced in athero-susceptible and athero-protected regions, respectively. However, markers for endoplasmic reticulum stress, a signature of susceptible endothelial phenotype, were not further enhanced by brief hypercholesterolemia. Both region-specific and ubiquitous (ABCA1) phenotype changes were identified as early pre-lesional responses of the endothelium to hypercholesterolemia.