Current clinical treatment for mercury poisoning generally depends on the complexation of mercury ions with dithiol compounds. Although mercury is known to have a high binding affinity for the soft sulfur donor atoms, further understanding of its complex formation tendencies in the presence of auxiliary donor atoms is necessary to enhance its immobilization and detoxification. In order to evaluate the effect of auxiliary binding groups on mercury complex formation, two dicysteinyl tripeptides, CXC, where C is cysteine and X is glycine (CGC) or glutamic acid (CEC) were reacted with mercury(II) chloride. These peptides provide the structural differences to evaluate the role of an added gamma carboxylate group in complex formation. The reaction mixtures were studied by Electrospray Ionization Mass Spectrometry (ESI-MS). Low micromolar mercury(II) solutions consisting of mercury(II) to peptide molar ratio of 1:2, 1:1, and 1:0.5 were analyzed for changes in complex speciation after mixing. The results show that the major complex formed is the 1:1 Hg(peptide) complex for both dithiol peptide ligands. Other complexes include 1:2 Hg(peptide)2, 2:2 Hg2(peptide)2, 2:3 Hg3(peptide)2 and 4:2 Hg4(peptide)2. But the CEC ligand also readily forms higher order peptide complexes such as 1:3 Hg(CEC)3, 2:3 Hg2(CEC)3, and 3:3 Hg3(CEC)3.Computational modeling using electronic structure methods was used to predict the structures and energetics of some candidate complexes. These calculations show that the thiol sulfur of the cysteine residues is the most important mercury coordination site. However, auxiliary binding groups including the carboxylate and amide groups in these peptides also play a significant role in stabilizing complexes through interaction with mercury(II) or hydrogen bonding.ESI-MS and computational data provide corroborating evidences that auxiliary donor atoms play an important role in mercury-dithiol complex formation and speciation. These results will be useful toward the rational design of dithiols for optimal mercury chelation.