Although S-nitrosothiols are regarded as important elements of many NO-dependent signal transduction pathways, the physiological mechanism of their formation remains elusive. Here, we demonstrate a novel mechanism by which cytochrome c may represent an efficient catalyst of S-nitrosation in vivo. In this mechanism, initial binding of glutathione to ferric cytochrome c is followed by reaction of NO with this complex, yielding ferrous cytochrome c and S-nitrosoglutathione (GSNO). We show that when submitochondrial particles or cell lysates are exposed to NO in the presence of cytochrome c, there is a robust formation of protein S-nitrosothiols. In the case of submitochondrial particles protein S-nitrosation is paralleled by an inhibition of mitochondrial complex I. These observations raise the possibility that cytochrome c is a mediator of S-nitrosation in biological systems, particularly during hypoxia, and that release of cytochrome c into the cytosol during apoptosis potentially releases a GSNO synthase activity that could modulate apoptotic signaling.