Sirtuin-1 (SIRT1) is an NAD(+)-dependent protein deacetylase that is sensitive to oxidative signals. Our purpose was to determine whether SIRT1 activity is sensitive to the low molecular weight nitrosothiol, S-nitrosoglutathione (GSNO), which can transduce oxidative signals into physiological responses. SIRT1 formed mixed disulfides with GSNO-Sepharose, and mass spectrometry identified several cysteines that are modified by GSNO, including Cys-67 which was S-glutathiolated. GSNO had no effect on basal SIRT1 deacetylase activity, but inhibited stimulation of activity by resveratrol (RSV) with an IC(50) of 69 microM. These observations indicate that S-glutathiolation of SIRT1 by low concentrations of reactive glutathione can modulate its enzymatic activity.