Objectives We investigated whether lowering of fasting homocysteine concentrations, either with folic acid or with betaine supplementation, differentially affects vascular function, a surrogate marker for risk of cardiovascular disease, in healthy volunteers. As yet, it remains uncertain whether a high concentration of homocysteine itself or whether a low folate status¿its main determinant¿is involved in the pathogenesis of cardiovascular disease. To shed light on this issue, we performed this study. Design This was a randomized, placebo-controlled, double-blind, crossover study. Setting The study was performed at Wageningen University in Wageningen, the Netherlands. Participants Participants were 39 apparently healthy men and women, aged 50¿70 y. Interventions Participants ingested 0.8 mg/d of folic acid, 6 g/d of betaine, and placebo for 6 wk each, with 6-wk washout in between. Outcome Measures At the end of each supplementation period, plasma homocysteine concentrations and flow-mediated dilation (FMD) of the brachial artery were measured in duplicate. Results Folic acid supplementation lowered fasting homocysteine by 20% (¿2.0 ¿mol/l, 95% confidence interval [CI]: ¿2.3; ¿1.6), and betaine supplementation lowered fasting plasma homocysteine by 12% (¿1.2 ¿mol/l; ¿1.6; ¿0.8) relative to placebo. Mean (± SD) FMD after placebo supplementation was 2.8 (± 1.8) FMD%. Supplementation with betaine or folic acid did not affect FMD relative to placebo; differences relative to placebo were ¿0.4 FMD% (95%CI, ¿1.2; 0.4) and ¿0.1 FMD% (¿0.9; 0.7), respectively. Conclusions Folic acid and betaine supplementation both did not improve vascular function in healthy volunteers, despite evident homocysteine lowering. This is in agreement with other studies in healthy participants, the majority of which also fail to find improved vascular function upon folic acid treatment. However, homocysteine or folate might of course affect cardiovascular disease risk through other mechanisms.