Nitric oxide (NO), a highly reactive gas, is now established as a major messenger molecule regulating blood vessel dilation, immune functions and serving as a neurotransmitter in brain and peripheral nervous system. NO can also act as a tumoricidal and bactericidal molecule. The effect of NO to dilate blood vessels is largely explained by stimulation of soluble guanylate cyclase (a heme-iron containing protein) leading to formation of cGMP and protein phosphorylation. This is considered to be the main physiological signaling mechanism of NO. NO also binds to non-heme iron-containing proteins and this has been considered as a pathophysiological or cytotoxic action of NO. Furthermore, NO, more correctly nitrosonium (NO+) which can be formed by the removal of one electron, reacts with protein SH-groups to cause the S-nitrosylation of proteins. We have recently established a link between NO and the S-nitrosylation and mono-ADP-ribosylation of the enzyme glyceraldehyde 3-monophosphate dehydrogenase, which adds a further protein modification mechanism for NO action. This links the formation of the second messenger molecule NO to post-translational protein modification and adds a new dimension to NO in the communication of intracellular signals.