Dinitrosyl iron complexes (DNICs) are ubiquitous in mammalian cells and tissues producing nitric oxide (NO) and have been argued to play key physiological and pathological roles. Nonetheless, the mechanism and dynamics of DNIC formation in aqueous media remain only partially understood. Here, we report a stopped-flow kinetics and density functional theory (DFT) investigation of the reaction of NO with ferrous ions and the low molecular weight thiols glutathione (GSH) and cysteine (CysSH) as well as the peptides WCGPC and WCGPY to produce DNICs in pH 7.4 aqueous media. With each thiol, a two-stage reaction pattern is observed. The first stage involves several rapidly established preequilibria leading to a ferrous intermediate concluded to have the composition F-II(NO)(RS)(2)(H2O)(x)(C). In the second stage, C undergoes rate-limiting, unimolecular autoreduction to give thiyl radical (RS center dot) plus the mononitrosyl Fe(I) complex Fe-I(NO)(RS)(H2O)v following the reactivity order of CysSH > W CGPC > W CGPY > GSH. Time course simulations using the experimentally determined kinetics parameters demonstrate that, at a NO flux characteristic of inflammation, DNICs will be rapidly formed from intracellular levels of ferrous iron and thiols. Furthermore, the proposed mechanism offers a novel pathway for S-nitroso thiol (RSNO) formation in a biological environment.