Background—Homocysteine is a risk factor for coronary artery disease (CAD), although a causal relation remains to be proven. The importance of determining direct causality rests in the fact that plasma homocysteine can be safely and inexpensively reduced by 25% with folic acid. This reduction is maximally achieved by doses of 0.4 mg/d. High-dose folic acid (5 mg/d) improves endothelial function in CAD, although the mechanism is controversial. It has been proposed that improvement occurs through reduction in total (tHcy) or free (non–protein bound) homocysteine (fHcy). We investigated the effects of folic acid on endothelial function before a change in homocysteine in patients with CAD.Methods and Results—A randomized, placebo-controlled study of folic acid (5 mg/d) for 6 weeks was undertaken in 33 patients. Endothelial function, assessed by flow-mediated dilatation (FMD), was measured before, at 2 and 4 hours after the first dose of folic acid, and after 6 weeks of treatment. Plasma folate increased markedly by 1 hour (200 compared with 25.8 nmol/L;P<0.001). FMD improved at 2 hours (83 compared with 47 &mgr;m;P<0.001) and was largely complete by 4 hours (101 compared with 51 &mgr;m;P<0.001). tHcy did not significantly differ acutely (4-hour tHcy, 9.56 compared with 9.79 &mgr;mol/L;P=NS). fHcy did not differ at 3 hours but was slightly reduced at 4 hours (1.55 compared with 1.78 &mgr;mol/L;P=0.02). FMD improvement did not correlate with reductions in either fHcy or tHcy at any time.Conclusions—These data suggest that folic acid improves endothelial function in CAD acutely by a mechanism largely independent of homocysteine.