The S-nitroso adducts of nitric oxide (NO) may serve as carriers of NO and play a role in cell signaling and/or cytotoxicity. A quantitative understanding of the kinetics of S-nitrosothiol formation in solutions containing NO and O₂ is important for understanding these roles of S-nitroso compounds in vivo. Rates of S-nitrosation in aqueous solutions were investigated for three thiols:  glutathione, N-acetylcysteine, and N-acetylpenicillamine. Nitrous anhydride (N₂O₃), an intermediate in the formation of nitrite from NO and O₂, is the most likely NO donor for N-nitrosation of amines as well as for S-nitrosation of thiols, at physiological pH. This motivated the use of a competitive kinetics approach, in which the rates of thiol nitrosation were compared with that of a secondary amine, morpholine. The kinetic studies were carried out with known amounts of NO and O₂ in solutions containing one thiol (400 μM) and morpholine (200−5700 μM) in 0.01 M phosphate buffer at pH 7.4 and 23 °C. It was found that disulfide formation, transnitrosation reactions, and decomposition of the S-nitrosothiol products were all negligible under these conditions. The rate of formation of S-nitrosothiols was expressed as k₇[N₂O₃][RSH], where RSH represents the thiol. The rate constant for S-nitrosation relative to that for N₂O₃ hydrolysis (k₄) was found to be k₇/k₄ = (4.15 ± 0.28) × 10⁴, (2.11 ± 0.11) × 10⁴, and (0.48 ± 0.04) × 10⁴ M^-1 for glutathione, N-acetylcysteine, and N-acetylpenicillamine, respectively. The overall (observed) rates of nitrosothiol formation reflect the fact that [N₂O₃] ∝ [NO]²[O₂] and that [N₂O₃] also depends on [RSH] and the concentration of phosphate. Using a detailed kinetic model to account for these effects, the present results could be reconciled with apparently dissimilar findings reported previously by others.