Folate supplementation improves endothelial function in patients with hyperhomocysteinemia. Mechanistic insights into potential benefits of folate on vascular function in general population however, remain mysterious. Expression of dihydrofolate reductase (DHFR) was markedly increased by folic acid (FA, 50 micromol/L, 24 h) treatment in endothelial cells. Tetrahydrofolate (THF) is formed after incubation of purified DHFR or cellular extracts with 50 micromol/L of substrate dihydrofolic acid. THF could then be detected and quantified by high performance liquid chromatography (HPLC) with a fluorescent detector (295/365 nm). Using this novel and sensitive assay, we found that DHFR activity was significantly increased by FA. Furthermore, FA improved redox status of Ang II treated cells by increasing H(4)B and NO() bioavailability while decreasing superoxide (O(2)(-)) production. It however failed to restore NO() levels in DHFR siRNA-transfected or methotrexate pre-treated cells, implicating a specific and intermediate role of DHFR. In mice orally administrated with FA (15 mg/kg/day, 16 days), endothelial upregulation of DHFR expression and activity occurred in correspondence to improved NO() and H(4)B bioavailability, and this was highly effective in reducing Ang II infusion (0.7 mg/kg/day, 14 days)-stimulated aortic O(2)(-) production. 5'-methyltetrahydrofolate (5'-MTHF) levels, GTPCH1 expression and activity remained unchanged in response to FA or Ang II treatment in vitro and in vivo. FA supplementation improves endothelial NO() bioavailability via upregulation of DHFR expression and activity, and protects endothelial cells from Ang II-provoked oxidant stress both in vitro and in vivo. These observations likely represent a novel mechanism (intermediate role of DHFR) whereby FA induces vascular protection.