Although different routes for the S-nitrosation of cysteinyl residues have been proposed, the main in vivo pathway is unknown. We recently demonstrated that direct (as opposed to autoxidation-mediated) aerobic nitrosation of glutathione is surprisingly efficient, especially in the presence of Mg2+. In the present study we investigated this reaction in greater detail. From the rates of NO decay and the yields of nitrosoglutathione (GSNO) we estimated values for the apparent rate constants of 8.9±0.4 and 0.55±0.06M−1s−1 in the presence and absence of Mg2+. The maximum yield of GSNO was close to 100% in the presence of Mg2+ but only about half as high in its absence. From this observation we conclude that, in the absence of Mg2+, nitrosation starts by formation of a complex between NO and O2, which then reacts with the thiol. Omission of superoxide dismutase (SOD) reduced by half the GSNO yield in the absence of Mg2+, demonstrating O2− formation. The reaction in the presence of Mg2+ seems to involve formation of a Mg2+•glutathione (GSH) complex. SOD did not affect Mg2+-stimulated nitrosation, suggesting that no O2− is formed in that reaction. Replacing GSH with other thiols revealed that reaction rates increased with the pKa of the thiol, suggesting that the nucleophilicity of the thiol is crucial for the reaction, but that the thiol need not be deprotonated. We propose that in cells Mg2+-stimulated NO/O2-induced nitrosothiol formation may be a physiologically relevant reaction.