Nitric-oxide synthase (NOS) catalyzes the five-electron oxidation of L-arginine to the nitric oxide radical (.NO) and L-citrulline. Molecular oxygen is the cosubstrate of the enzyme. NO synthase activity has been found in a large variety of cells and tissues. The enzyme exists in several isoforms, three of which have been purified, characterized, and cloned. The activities of all three isoforms are found distributed between the soluble and particulate fractions of cells. Isoform I (from brain) and isoform II (from cytokine-induced macrophages) are mostly soluble proteins. Isoform III from endothelial cells is myristoylated and found predominantly in the particulate fraction. The activities of isoforms I and III are regulated by Ca2+ in the nanomolar range. The activity of isoform II is Ca2+ independent. Similar, but not identical procedures are used to purify the different isozymes. All three isoforms are hemoproteins and require the same cofactors, NADPH (6R)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN). Flavins and tetrahydrobiopterin are found bound to the purified enzymes in quantities that are sometimes, but not always, sufficient for full activity. This chapter describes the methods for determining the flavin and biopterin content of NO synthase. © 1994, Elsevier Inc. All rights reserved.