GTP cyclohydrolase 1 is the rate-limiting enzyme in the production of tetrahydrobiopterin, a necessary cofactor for endothelial nitric oxide synthase. We tested the hypothesis that inhibition of tetrahydrobiopterin synthesis will impair endothelium-dependent relaxation and increase blood pressure in rats. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a GTP cyclohydrolase 1 inhibitor, was given in the drinking water (~120 mg/kg/day) of male Sprague-Dawley rats for 3 days. Systolic blood pressures were measured (tail-cuff procedure) for 3 days prior to and each day during DAHP treatment. Blood pressure was significantly increased following DAHP treatment (mmHg: 122±2 versus 154±3 prior to and following DAHP treatment, respectively; p<0.05). Endothelium-intact aortic segments from sodium pentobarbitol-anesthetized rats were isolated and hung in organ chambers for measurement of isometric force generation. Aortas from DAHP-treated rats exhibited a decreased maximal relaxation to acetylcholine compared to controls (% relaxation from phenylephrine-induced contraction 10 ^-7 mol/L: DAHP=57±6% versus control=79±4%; p<0.05). Relaxation responses to A23187 were also decreased in aortas from DAHP-treated rats compared to controls. Incubation with sepiapterin (10 ^-4 mol/L, 1 hour), which produces tetrahydrobiopterin via a salvage pathway, restored relaxation to acetylcholine in aortas from DAHP-treated rats. Superoxide dismutase significantly increased ACH-induced relaxation in aortas from DAHP-treated rats, whereas catalase had no effect. Endothelium-independent relaxation to sodium nitroprusside in aortas from DAHP-treated rats was not different from control rats, however, nitric oxide synthase inhibition increased sensitivity to sodium nitroprusside in aortas from DAHP-treated rats. These results support the hypothesis that GTP cyclohydrolase 1 inhibition decreases relaxation and increases blood pressure in rats.