During the last two decades nitric oxide (NO) has emerged as a new chemical messenger in plant biology, which is involved in many different physiological processes, such as plant defense, transpiration and gas exchange, seed germination, and root development. Protein S-nitrosylation, the post-translational modification of thiol residues, has been suggested to be the most important mechanism for transduction of the bioactivity of NO. The characterization of protein S-nitrosylation as well as the physiological relevance of this type of modification is essential information, which is necessary to understand the function of NO in plants. In this review we focus on the formation of nitrosothiols and describe the chemistry of NO and thiol groups. Furthermore, different methods for detection of S-nitrosothiols are highlighted and the function of S-nitrosylation in plants is discussed.