The characteristics of tetrahydrobiopterin (H₄biopterin) binding to pteridine-free recombinant macrophage inducible nitric oxide synthase expressed in Escherichia coli were investigated with a special focus given to effects caused by 2,4-diamino-5,6,7,8-tetrahydro-6-(l-erythro-1,2-dihydroxypropyl)pteridine (4-amino-H₄biopterin), a novel pterin-based inhibitor of nitric oxide synthase. The 4-amino compound completely inhibited enzyme stimulation by 10 μM H₄biopterin with a half-maximally active concentration of 7.2 ± 0.39 μM, whereas H₂biopterin and sepiapterin were much less potent. Binding studies using [³H]H₄biopterin at 4 °C revealed biphasic association of the radioligand according to two first-order reactions with apparent rate constants of 2.2 and 0.05 min^-1, each accounting for approximately 50% of total binding. Dissociation of [³H]H₄biopterin occurred with rate constants of 0.005 and 0.0028 min^-1 in the absence and presence of l-arginine, respectively. Specific binding of 10 nM [³H]H₄biopterin was antagonized by unlabeled H₄biopterin and its 4-amino analog with half-maximal effects at 84 ± 6 and 34 ± 3.2 nM, respectively. Binding of H₄biopterin and 4-amino-H₄biopterin was accompanied by a partial low spin to high spin conversion of the heme that was completed by l-arginine. Similarly, the active cofactor and the inhibitory 4-amino derivative both induced significant formation of stable protein dimers that survived during SDS electrophoresis, suggesting that the allosteric effects caused by H₄biopterin do not explain sufficiently the essential role of the pteridine cofactor in NO biosynthesis.