Inhibition of GTP cyclohydrolase I (GTPCH) has been used as a selective tool to assess the role of de novo synthesis of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) in a biological system. Toward this end, 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used as the prototypical GTPCH inhibitor. Using a novel real-time kinetic microplate assay for GTPCH activity and purified prokaryote-expressed recombinant proteins, we show that potent inhibition by DAHP is not the result of a direct interaction with GTPCH. Rather, inhibition by DAHP in phosphate buffer occurs via an indirect mechanism that requires the presence of GTPCH feedback regulatory protein (GFRP). Notably, GFRP was previously discovered as the essential factor that reconstitutes inhibition of pure recombinant GTPCH by the pathway end product BH4. Thus, DAHP inhibits GTPCH by engaging the endogenous feedback inhibitory system. We further demonstrate that L-Phe fully reverses the inhibition of GTPCH by DAHP/GFRP, which is also a feature in common with inhibition by BH4/GFRP. These findings suggest that DAHP is not an indiscriminate inhibitor of GTPCH in biological systems; instead, it is predicted to preferentially attenuate GTPCH activity in cells that most abundantly express GFRP and/or contain the lowest levels of L-Phe.(6R)-5,6,7,8-Tetrahydro-L-biopterin (BH4) 1 is an essential cofactor for phenylalanine hydroxylase (1), tyrosine hydroxylase (2), tryptophan hydroxylase (3), glycerol ether monoxygenases (4, 5), and the three isoforms of nitric-oxide synthase (6, 7). Because the activity of these enzymes is typically limited by the availability of BH4, intracellular levels of BH4 determine the rate of production of several key cell-signaling molecules including nitric oxide (NO), dopamine, norepinephrine, epinephrine, and serotonin. In mammalian cells, BH4 levels are primarily dictated by the activity of GTP cyclohydrolase I (GTPCH), the first of three enzymes in the de novo BH4 synthesis pathway (8). Mutations in the GTPCH gene are responsible for severe diseases including 3,4-dehydroxyphenylalanine (dopa)-responsive dystonia (9) and atypical phenylketonuria (10). Other neurological disorders, including Parkinson's disease (11), Alzheimer's disease (12, 13), depression (14), and vascular endothelial dysfunction resulting from diabetes (15, 16), smoking (17-19), and hypercholesterolemia (20), are all associated with deficient BH4 levels. The prototypical GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used extensively to assess the roles of BH4 and GTPCH in physiological and pathophysiological conditions, although the exact mechanism of this inhibition has not been defined.Four decades ago, DAHP was identified as a potent inhibitor of biopterin-dependent growth of the protozoan Crithidia fasciculata (21). Investigations of biopterin excretion in rats suggested the presence of a de novo biopterin synthesis pathway that is potently inhibited following DAHP treatment (22). Subsequently, biosynthesis of 7,8-dihydrobiopterin from GTP was demons...