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Inducible nitric oxide synthase (NOS) is a cytochrome P450-type hemoprotein that catalyzes the oxidation of scL-arginine to citrulline and nitric oxide ($\cdot$NO). The reaction requires NADPH and $\rm O\sb2$ and proceeds via the intermediate $N\sp{\rm G}$-hydroxy- scL-arginine (NHA). The enzyme binds an equivalent of FAD, FMN, iron protoporphyrin IX heme and 6(R)-tetrahydro- scL-biopterin $\rm(H\sb4B)$ per subunit. Calmodulin is tightly bound and copurifies with the enzyme. The role of $\rm H\sb4B$ in NOS has remained one of the least understood questions with respect to the mechanism of this reaction. Although NOS was thought to function as a self-sufficient cytochrome P450 hemoprotein, the effects of $\rm H\sb4B$ on enzyme stability, activity, oligomeric state and heme spin-state point to an important and perhaps multifunctional role for the reduced pterin. Like other P450 enzymes, the addition of substrate shifts the NOS heme spin-state from low- to high-spin. However, $\rm H\sb4B$ was also shown to influence the heme spin-state. $\rm H\sb4B$-free NOS is low-spin, and consists of a mixture of monomers and homodimers. Reconstitution of $\rm H\sb4B$-free NOS with $\rm H\sb4B$ resulted in high-spin, dimeric NOS. $\rm H\sb4B$ reconstitution was greatly facilitated by the presence of a thiol. Substitution of the redox-inactive, $6(R,S)$-methyl-5-deazatetrahydropterin $\rm(DZPH\sb4)$ for $\rm H\sb4B$ did not support NOS activity, nor stabilize or induce dimerization. However, $\rm DZPH\sb4$ did result in the rapid, thiol-independent formation of high-spin heme. Additionally, $\rm H\sb4B$-free NOS catalyzed the oxidation of NHA with either hydrogen peroxide $\rm(H\sb2O\sb2)$ or NADPH and $\rm O\sb2$ as substrates. No product formation from arginine was observed under either condition. The amino acid products of NHA oxidation in both $\rm H\sb2O\sb2$ and $\rm NADPH/O\sb2$ reactions were determined to be citrulline and $N\sp\delta$-cyanoornithine. The inorganic product of the $\rm NADPH/O\sb2$ reaction is believed to be nitroxyl anion. Reconstitution of $\rm H\sb4B$-free NOS with $\rm H\sb4B$ resulted in reconstitution of $\cdot$NO-forming activity. Thus, the 6-dihydroxypropyl side chain of the reduced biopterin appears to be an important factor in NOS dimerization, and the pteridine ring influences the heme spin equilibrium. The redox activity of $\rm H\sb4B$ is necessary for $\cdot$NO formation and is postulated to function in the hydroxylation of arginine to NHA. Such a reaction has precedent in the reaction catalyzed by phenylalanine hydroxylase. $\rm H\sb4B$ also seems to play a role in the oxidation of NHA to citrulline and $\cdot$NO by either direct participation or modulation of the electron transfer pathways of NOS.