Decreased levels of tetrahydrobiopterin (BH4), an absolute cofactor for nitric oxide synthase (NOS), lead to uncoupling of NOS into a superoxide v. nitric oxide producing enzyme, and it is this uncoupling that links it to the development of vascular disease. However, the effects of in vivo deficiency of BH4 on neointimal formation after vascular injury have not been previously investigated. Hph-1 mice, which display 90% deficiency in guanine triphosphate cyclohydrolase I, the rate limiting enzyme in BH4 synthesis, were used. Hph-1 and wild-type mice, treated with either vehicle or BH4 ( n = 15 per group), were subjected to wire-induced femoral artery injury, and NOS expression and activity, inflammation, cell proliferation, superoxide production, and neointimal formation were assessed. The major form of NOS expressed over vessel wall after vascular injury was endothelial NOS. Hph-1 mice exhibited lower NOS activity (2.8 ± 0.3 vs. 4.5 ± 0.4 pmol/min/mg protein, P < 0.01), and higher aortic superoxide content (5.2 ± 2.0 × 10⁵ cpm vs. 1.6 ± 0.7 × 10⁵ cpm, P < 0.01) compared with wild-type controls, indicating uncoupling of NOS. Treatment of hph-1 mice with BH4 significantly increased NOS activity (from 2.8 ± 0.3 to 4.1 ± 0.4 pmol·min⁻¹·mg protein⁻¹, P < 0.05), and attenuated superoxide production (from 5.2 ± 2.0 × 10⁵ cpm to 0.8 ± 0.7 × 10⁵ cpm, P < 0.05). Hph-1 mice also had higher inflammatory reactions and more cell proliferation after vascular injury. Furthermore, hph-1 mice responded by a marked increase in neointimal formation at 4 wk after vascular injury, compared with wild-type controls (intima:media ratio: 4.5 ± 0.5 vs. wild-type 0.7 ± 0.1, P < 0.001). Treatment of hph-1 mice with BH4 prevented vascular injury-induced increase in neointimal formation (intima:media ratio: 1.4 ± 0.1 vs. hph-1, P < 0.001). Treatment had no effect on wild-type controls. In summary, we describe, for the first time, that in vivo BH4 deficiency facilitates neointimal formation after vascular injury. Modulation of BH4 bioavailability is an important therapeutic target for restenosis.