Acute inflammation causes endothelial dysfunction, which is partly mediated by oxidant stress and inactivation of nitric oxide (NO). The contribution of depletion of tetrahydrobiopterin (BH4), the co-factor required for NO generation, is unclear. In this randomized, double blind, three-way cross-over study, forearm blood flow (FBF) responses to acetylcholine (ACh) and glyceryltrinitrate (GTN) were measured before and 3.5 hours after administration of Escherichia coli endotoxin (LPS, 20 IU/kg i.v.) in 8 healthy males. The effect of intra-arterial BH4 (500 µg/min), placebo, or vitamin C (24 mg/min) was studied on separate study days 3.5 hours after LPS. Additionally human umbilical cord vein endothelial cells (HUVEC) were incubated with vitamin C and LPS for 24 hours. ACh and GTN caused dose-dependent forearm vasodilation. LPS decreased FBF responses to ACh by 23 ± 17 % (P<0.05), which was restored to baseline reactivity by BH4 and vitamin C. FBF responses to GTN were not affected by BH4 or vitamin C. LPS increased leukocyte count, high sensitivity C-reactive protein, interleukin (IL)-6, IL-1 beta, interferon gamma, monocyte chemoattractant protein-1, pulse rate and body temperature and decreased platelet count and vitamin C concentrations. Vitamin C increased forearm plasma concentrations of BH4 by 32 % (P<0.02). HUVEC intracellular BH4 concentrations increased when incubated with LPS and vitamin C but not with LPS alone. Impaired endothelial function during acute inflammation can be restored by BH4 or vitamin C. Vitamin C may exert some of its salutary effects by increasing BH4 concentrations.