Pathological nitric oxide (NO) generation in sepsis, inflammation and stroke may be therapeutically controlled by inhibiting NO synthases (NOS). Here we targeted the (6R)-5,6,7,8-tetrahydro-L-biopterin (H₄Bip) binding site of NOS, which, upon cofactor binding, maximally increases enzyme activity and NO production from substrate L-arginine. The first generation of H₄Bip-based NOS inhibitors employed a 4-amino pharmacophore of H4Bip analogous to anti-folates such as methotrexate. Here we developed a novel series of 4-oxo pteridine derivatives that were screened for inhibition against neuronal NOS (NOS-I) and a structure-activity relationship was determined. In order to understand the structural basis for pterin antagonism, selected derivatives were docked into the NOS pterin binding cavity. Using a reduced 4-oxo pteridine-scaffold, derivatives with certain modifications such as electron-rich aromatic phenyl or benzoyl groups at the 5- and 6-positions, were discovered to markedly inhibit NOS-I, possibly due to hydrophobic and electrostatic interactions with Phe⁴⁶² and Ser¹⁰⁴, respectively, within the pterin binding pocket. The favorable effect of N-5 benzoyl substitution was counteracted by additional steric bulk in the 6- and 7-positions revealing steric hindrance, possibly with Ser¹⁰⁴ and Phe⁴⁶². One of the most effective 4-oxo compounds and, for comparisons an active 4-amino derivative, were then co-crystallized with the endothelial NOS (NOS-III) oxygenase domain and this structure solved in order to confirm the hypothetical binding modes. Collectively, these findings suggest (i) that, unlike the anti-folate principle, the 4-amino substituent is not essential for developing pterin-based NOS inhibitors and (ii), provide a steric and electrostatic basis for their rational design.