Moderate elevation of plasma homocyst(e)ine is associated with increased risk for atherosclerotic vascular disease. In a previous study, we observed impaired vascular function in nonatherosclerotic monkeys with moderate hyperhomocyst(e)inemia. In this study, we tested the hypothesis that dietary intervention to lower plasma homocyst(e)ine corrects vascular dysfunction in atherosclerotic monkeys. Cynomolgus monkeys were fed an atherogenic diet that produces both hypercholesterolemia and moderate hyperhomocyst(e)inemia. After 17 months, the atherogenic diet was supplemented with B vitamins (5 mg folic acid, 400 μg vitamin B-12, and 20 mg vitamin B-6 daily) for 6 months. Total plasma homocyst(e)ine decreased from 12.8±2.8 to 3.5±0.3 μmol/L (n=9; mean±SE; P<.01) after vitamins were added to the diet, but plasma cholesterol remained elevated (522±63 versus 514±41 mg/dL; P>.05). In response to intra-arterial infusion of collagen, blood flow to the leg decreased by 30±3% and 38±5%, respectively, before and after vitamin supplementation (P>.05). In vivo responses of resistance vessels to endothelium-dependent vasodilators (acetylcholine or ADP) were impaired at baseline and did not improve after vitamin supplementation. In carotid artery studied ex vivo, relaxation to low doses of acetylcholine improved after vitamin supplementation, but maximal relaxation remained impaired. Ex vivo thrombomodulin anticoagulant activity was threefold higher in monkeys fed the atherogenic diet (with or without B vitamins) than in normal monkeys (P<.05). We conclude that normalization of plasma homocyst(e)ine is insufficient to restore normal vascular function in atherosclerotic monkeys with persistent hypercholesterolemia and that atherosclerosis, with or without hyperhomocyst(e)inemia, is associated with elevated thrombomodulin activity.