S-nitros(yl)ation belongs to the redox-based posttranslational modifications of proteins but the underlying chemistry is controversial. In contrast to current concepts involving the autoxidation of nitric oxide ((.)NO, nitrogen monoxide), we and others have proposed the formation of peroxynitrite (oxoperoxonitrate (1(-))as an essential intermediate. This requires low cellular fluxes of (.)NO and superoxide (UO2(-)), for which model systems have been introduced. We here propose two new systems for nitros(yl)ation that avoid the shortcomings of previous models. Based on the thermal decomposition of 3-morpholinosydnonimine,equal fluxes of (.)NO and UO2(-) were generated and modulated by the addition of (.)NO donors or Cu,Zn superoxide dismutase. As reactants for S-nitros(yl)ation, NADP+-dependent isocitrate dehydrogenase and glutathione were employed, for which optimal S-nitros(yl)ation was observed at nanomolar fluxes of (.)NO and UO2(-) at a ratio of about 3:1. The previously used reactants phenol and diaminonaphthalene (C- and Nnitrosation)demonstrated potential participation of multiple pathways for nitros(yl)ation. According to our data, neither peroxynitrite nor autoxidation of UNO was as efficient as the 3 (.)NO/1 UO2(-) system in mediating S-nitros(yl)ation. In theory this could lead to an elusive nitrosonium (nitrosyl cation)-like species in the first step and to N2O3 in the subsequent reaction. Which of these two species or whether both together will participate in biological S-nitros(yl)ation remains to be elucidated. Finally, we developed several hypothetical scenarios to which the described (.)NO/UO2-flux model could apply, providing conditions that allow either direct electrophilic substitution at a thiolate or S-nitros(yl)ation via transnitrosation from S-nitrosoglutathione.