The biological effects of nitric oxide (NO) are in significant part mediated through S-nitrosylation of cysteine thiol. Work on model thiol substrates has raised the idea that molecular oxygen (O(2)) is required for S-nitrosylation by NO; however, the relevance of this mechanism at the low physiological pO(2) of tissues is unclear. Here we have used a proteomic approach to study S-nitrosylation reactions in situ. We identify endogenously S-nitrosylated proteins in subcellular organelles, including dihydrolipoamide dehydrogenase and catalase, and show that these, as well as hydroxymethylglutaryl-CoA synthase and sarcosine dehydrogenase (SarDH), are S-nitrosylated by NO under strictly anaerobic conditions. S-Nitrosylation of SarDH by NO is best rationalized by a novel mechanism involving the covalently bound flavin of the enzyme. We also identify a set of mitochondrial proteins that can be S-nitrosylated through multiple reaction channels, including anaerobic/oxidative, NO/O(2), and GSNO-mediated transnitrosation. Finally, we demonstrate that steady state levels of S-nitrosylation are higher in mitochondrial extracts than the intact organelles, suggesting the importance of denitrosylation reactions. Collectively, our results provide new insight into the determinants of S-nitrosothiol levels in subcellular compartments.