Nitric oxide (NO) was the first molecule afforded the status of gasotransmitter or gaseous signaling molecule. The seminal work by Furchgott, Ignarro, and Murad established the vasoactive functions of NO as an endothelial-derived relaxation factor. This was followed by the identification and cloning of its biosynthetic enzymes and elucidation of its role as a signaling molecule both in peripheral tissues and in the brain, where it functions as a neurotransmitter. NO is synthesized endogenously from the amino acid arginine. In this chapter, we review the biogenesis of NO and its regulation at multiple levels.