Nitric oxide (NO) is a very simple molecule that performs different biological functions, both in the intra- and extracellular space. This molecule is generated from the amino acid L-arginine, by the action of the nitric oxide synthase (NOS), enzymes which, in the presence of oxygen produces L-citrulin and nitric oxide. These enzymes require three substrates for their action: arginin, NADPH, and oxygen and five cofactors: haem group, tetrahydrobiopterin, calmodulin, FMN (flavin mononucleotide), and FAD (flavin adenine dinucleotide). The study of the metabolism of NO could be performed using inhibitors of the NOS. The main inhibitors used in the practice are analogues of arginine, such as L-NAME (N-nitroarginin methyl ester) and L-canavanin with an irreversible effect and L-NMMA (-monomethyl-Larginine) with a reversible action. Four types of NOS are described: neuronal NOS (nNOS), endothelial NOS (eNOS), mitochondrial NOS (mNOS), and inducible NOS (iNOS). Constitutive enzymes (nNOS and eNOS) requires for activation a calcium dependent union to calmodulin. In the case of iNOS, the union between the enzyme and calmodulin is not calcium dependent. The synthesis of iNOS is stimulated by bacterial substances, for example, lipopolysaccharide (LPS) and by different cytokines released by macrophages or Th1 lymphocytes. The main physiological actions of NO are (i) the control of vascular tone (arterial vasodilation and inhibition of adhesion and aggregation of platelets), (ii) neurotransmission (learning and memory at the central nervous system and relaxation of visceral smooth muscle in the peripheral nervous system), and (iii) pathogenesis and control of infectious and parasitic diseases. NO can be measured directly or indirectly. The direct type of measurement is difficult to perform, since it is a molecule with a very short half life and rapidly diffuses to the tissues to perform its action. The indirect methods more employed for the NO detection are Griess technique and NOS expression. Nitrites and/or nitrates can be detected as products of its metabolism by Griess technique. NOS detection can be performed by immunocytochemistry or Western Blot and gene expression by RT-PCR or real-time PCR.