Nitric oxide signaling is achieved through cGMP-dependent and-independent mechanisms. The latter are exemplified by the NAD (+)-dependent automodification of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The experimental post-translational, covalent modification of the enzyme by [32P] NAD+ is achieved using NO-releasing compounds and an active constitutive or inducible NO-synthase. Potential roles for NO in this covalent enzyme modification can be grouped as follows: S-Nitrosylation of GAPDH by NO+ NAD (+)-dependent, post-translational covalent automodification of GAPDH. Oxidative modification of GAPDH by NO-related compounds, probably ONOO. GAPDH modification by one of the proposed mechanisms would lead to inhibition of enzyme catalysis. It is likely that the NAD (+)-dependent automodification process occurs in vitro, in intact cells, and in whole animals. Besides its normal function in glycolysis, GAPDH not only is a target for NO-mediated direct and indirect modifications but also is ADP-ribosylated in the presence of brefeldin A (90). The relation of such ADP-ribosylation to enzyme activity is so far unknown. GAPDH also may be involved in one of the following functions unrelated to its glycolytic activity (81 and refs. therein; 90): binding and transport of tRNA associated with nuclear localization of GAPDH. DNA-repair activity, ie, uracil DNA glycosylase. Activation of transcription in neurons. Interaction with tubulin and microtubules. The transport of nitric oxide. Serves as a substrate for brefeldin A stimulated ADP-ribosylation. Because some of these alternative functions of GAPDH, just like NO-mediated modification of the enzyme, are related to the NAD+ binding site of the protein, we are interested in searching for the significance of these activities in relation to NO actions. In recent years, several functions of NO have been linked to direct, cGMP-independent actions. Modification of GAPDH is probably just one interesting target related to NO-redox chemistry and active-site thiol modification. It will be challenging to investigate NO biochemistry in closer detail and to elucidate how NO targets biological systems, especially in relation to the patho-physiological role of NO in medically related conditions.