Our group, as well as many others, has reported that a classic glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multiple function protein. We have reported that oxidized GAPDH may play a role in cytotoxicity associated with its translocation to the nucleus. We have also found in vivo ADP-ribosylated GAPDH in the nucleus upon oxidative stress, probably following an initial S-nitrosylation (Hara et al SFN 2002). Here we report roles of two cysteine residues, cysteine-150 and -154 in oxidative modification of GAPDH. These two cysteine residues are well conserved among many species. To analyze their function, we have produced three kinds of GAPDH mutants, GAPDH-C150S, GAPDH-C154S, and GAPDH-C150S/C154S (double mutant) in which each cysteine residue is substituted by serine. GAPDH was ADP-ribosylated in vitro by the mixture with 32P-labeled NAD under oxidative stress. We found no ADP-ribosylation in GAPDH-C150S, which fit with the current consensus that ADP-ribosylation occurs at cystein...