The physiological roles of insulin and nitric oxide (NO) have recently been recognized by several studies. A diversity of chemical modifications of insulin is reported both in vivo and in vitro. S-nitrosation, the covalent linkage of NO to cysteine-free thiol, is recognized as an important post-translational regulation in many proteins. Here, we report the in vitro synthesis of a S-nitrosothiol of bovine insulin A- and B-chains. These compounds were characterized by their HPLC chromatographic behavior, monitored by UV visible spectroscopy and electron spray ionization mass spectrometry. The experimental results indicate that each A- and B-chain were S-nitrosated with only one NO group. Stability and solubility of these synthesized derivatives is described for physiological purposes. In this work, nitroso A- and B-chains of insulin were synthesized in vitro in order to better understand the possible interactions between insulin and NO that may be involved in the etiology of insulin resistance.