Nitric oxide was recently demonstrated to stimulate ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Our studies on the effect of glyceraldehyde-3-phosphate (GA3P), the natural substrate of dehydrogenase activity of GAPDH, indicated GA3P to be another very potent activator of ADP-ribosylation of the enzyme. GA3P was able to activate ADP-ribosylation only in the presence of DTT. The action of GA3P was associated with inhibition of GAPDH dehydrogenase activity. Ka for GA3P was at least 50-fold lower and maximal activation was somewhat higher than these values for other aldehydes that were also able to enhance GAPDH ADP-ribosylation in the presence of DTT. ADP-ribosylation was blocked by carboxamidomethylation of the essential cysteine SH-group. The bond between the prelabeled protein and ADP-ribose was resistant to hydrolysis with hydroxylamine and HgCl2, suggesting that a lysine epsilon-amino group is the target for ADP-ribosylation.