Tetrahydrobiopterin (BH4) is an essential cofactor for all isoforms of nitric oxide synthase. While it is well established that BH4 deficiency states are associated with impairment of dopamine, serotonin and phenylalanine metabolism, less is known with regard to the effects of deficiency of the cofactor upon nitric oxide (NO) metabolism. In this study, we have evaluated the effects of partial BH4 deficiency upon (a) tissue availability of the antioxidant glutathione, (b) basal NO production and (c) NO generation following exposure to lipopolysaccharide (LPS), which is known to increase expression of the inducible form of nitric oxide synthase. Using the hph-1 mouse, which displays a partial BH4 deficiency owing to impaired activity of GTP cyclohydrolase, we report decreased levels of glutathione in brain and kidney and evidence for decreased basal generation of nitric oxide in the periphery (as judged by the plasma nitrate plus nitrite concentration). Following LPS administration, peripheral NO generation increases. However, the concentration of plasma nitrate plus nitrite achieved was significantly decreased in the hph-1 mouse. Furthermore, LPS administration caused loss of glutathione in both wild-type and hph-1 liver and kidney. It is concluded that cofactor replacement, sufficient to fully correct a cellular BH4 deficiency, may be of benefit to patients with inborn errors of BH4 metabolism.