Nitric oxide (NO), first recognized for its importance as an endothelium-dependent vasodilator, is now recognized as an important messenger molecule for the activation of soluble guanylate cyclase in a wide variety of tissues, including the central and the peripheral nervous system. In addition, NO by itself may have multiple functions independent of cyclic GMP, ranging from cytotoxicity to ADP ribosylation. In brain tissue, NO is produced enzymatically from l-arginine by NO synthase, a constitutive, calcium-, calmodulin-, and NADPH-dependent enzyme. In this article we describe several methods for the quantitation of NO synthase activity based on the measurement of l-citrulline, the stable by-product of NO synthesis from l-arginine. This method is sensitive, rapid, and reliable and can be performed with a simple experimental setup that is convenient for quantitating NO production of a large number of samples at a time. The l-citrulline methodology is compared to several other methods of quantifying NO and NO synthase activity, and its advantages and disadvantages are discussed.