Unlike the formation of nitrosothiols by nitrous acid, our study revealed that NO₂^- effectively reacted with L-cysteine or reduced glutathione (GSH) at pH 7.0 and 7.4, to form orange-pink products of S-nitrosocysteine (CySNO) or S-nitrosoglutathione (GSNO). The reactions were in a concentration-dependant manner. These products exhibited not only peak absorbances at around 340 and 540nm, but also unique colors and patterns of mobility on cellulose thin layer chromatographic plates. In comparison, the S-nitrosation of dithiothreitol was noted exclusively under acidic pH. In addition, the S-nitrosation of hemoglobin (Hb) by either peroxynitrite (PN) or NO₂^- at pH 6.0 was detected via Western blot. The half-life of degradation of CySNO in NO₂^- solution was significantly shorter than that of GSNO at a wide range of pH. In the absence of NO₂^-, degradation of GSNO was facilitated by incubation with L-cysteine, but not L-serine. In the signaling process involving NO ® PN ® NO₂^- ® CySNO/GSNO ® NO, L-cysteine may function as a NO-carrier to reach shorter-distance targets, and also an "activator" to release NO from GSNO. Furthermore, L-cysteine may play a vital role in reducing (severe) oxidative stress.