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Maternal exposure to Hg(II) during pregnancy has been identified as a potential causal factor in the development of severe neurobehavioral disorders. Children with autism have been identified with lower reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios, and GSH is known to strongly bind Hg(II). In order to gain insight into the mechanism by which GSH binds Hg(II), high resolution mass spectrometry coupled with tandem mass spectrometry was utilized to examine the conjugation process. While the 1:1 Hg(II):GSH conjugate is not formed immediately upon mixing aqueous solutions of Hg(II) and GSH, two species containing Hg(II) are observed:the 1:2 Hg(II):GSH conjugate, [(GS)2 Hg + H+], and a second Hg(II)-containing species around m/z 544. Interestingly, this species at m/z 544 decreases in time while the presence of the 1:1 Hg(II):GSH conjugate increases, suggesting that m/z 544 is an intermediate in the formation of the 1:1 conjugate. Experiments using the high mass accuracy capability of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry coupled to an electrospray ionization source indicate that the intermediate species is [GSH + HgCl]+, andnotthe 1:1 conjugate [Hg(GSH) – H + 2H2O]+postulated in previous literature. Further confirmation of [GSH + HgCl]+ is supported by collisionofinduced dissociation experiments, which show neutral loss of HCl from the intermediate and loss of the N- and C-terminal amino acids, indicating binding of Hg(II) at the Cys residue.