High levels of plasma low-density lipoproteins (LDL) are known to be a risk factor for developing coronary artery disease although the specific mechanism involved is unknown. It may be related to effects of oxidized lipid components of LDL on vascular endothelial barrier function (EBF). This study addressed the hypothesis that LDL-associated products of cholesterol oxidation, oxysterols, decrease EBF resulting in increased penetration of blood components such as LDL into the arterial wall. LDL from human volunteers and rabbits was enriched with cholesterol or cholestan-3β, 5α, 6βtriol (Triol) by in vitro incubation. Exposure of cultured vascular endothelial cell monolayers to LDL enriched with Triol reduced EBF, measured as an increase in transendothelial albumin transfer, whereas cholesterol enrichment, like un-enriched LDL, had no effect on EBF. In a second experimental series, rabbits were gavaged with 100 mg of cholesterol or Triol/kg body weight, and LDL was isolated from serum 24 h after gavage. As was seen with the in vitro experiments, Triol-enriched LDL markedly decreased EBF. Similarly, LDL from cholesterol-gavaged rabbits reduced EBF, while LDL from vehicle treated rabbits had no effect. These results suggest that LDL-associated oxysterols are detrimental to normal barrier function of the vascular endothelium. Disruption of this barrier function may serve as an initiating factor in atherosclerotic lesion formation.