Chemical oxidants can induce the covalent binding of low molecular weight thiols to reactive sulfhydryls on proteins (S-thiolation). We found that stimulation of the respiratory burst of human blood monocytes resulted in S-thiolation of several proteins, most prominently one of 38 kDa. This purified protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by enzyme activity, immunoblotting, and amino acid analysis. After stimulation of the respiratory burst, S-thiolation of GAPDH gradually increased, and cytosol GAPDH activity decreased; so that at 60 min, GAPDH activity was reduced by approximately 40%. Activity was restored by the addition of the sulfhydryl-reducing agent dithioerythritol. H2O2 appeared to be particularly important in mediating S-thiolation during the respiratory burst. Exposure of monocytes to H2O2 induced concentration-dependent S-thiolation of GAPDH and a concomitant decrease in enzyme activity. The addition of respiratory burst stimuli to lymphocytes, which lack a full respiratory burst, had no effect on GAPDH S-thiolation or activity; but H2O2 induced S-thiolation of lymphocyte GAPDH and inhibition of enzyme activity. Stimulation of monocytes from three patients with chronic granulomatous disease resulted in no respiratory burst, S-thiolation of GAPDH, or inactivation of GAPDH activity. The thiols covalently bound to purified S-thiolated GAPDH were removed by dithioerythritol and were identified as glutathione and cysteine; glutathione was predominant. These results indicate that during the respiratory burst in monocytes, low molecular weight thiols can bind to specific cytosolic proteins, including GAPDH. It is possible that S-thiolation of cytosolic proteins serves to modulate cellular metabolic events during phagocytosis.