The gas-phase binding chemistry between Ca2+, Co2+, and Ni2+ and 33 tri- through decapeptides is evaluated with respect to aqueous-phase and theoretical chemistry. Metastable ion decompositions of tetrapeptide and larger peptide complexes that contain either hydrocarbon amino acid side chains or Pro reveal the intrinsic binding preferences. Tripeptide complexes do not. Interactions with the Ca2+-specific binding sequence in staphylococcal nuclease and with the Asp side chain in the third position from the N-terminus are also presented. For Co2+ and Ni2+, aqueous-phase chemistry is reflected in the gas-phase results. Mass spectrometry detects some weakly abundant, less favorable complexes, species that aqueous-phase studies do not detect. Gas-phase complexes of Ca2+ are not seen in solution because aqueous equilibria favor precipitation of Ca(OH)(2). Solution-phase binding interactions between Co2+ and Ni2+, however, are intrinsic, independent of solvation. Metastable ion decomposition mass spectrometry has a future in the direct elucidation of important metal ion binding sites in peptides and proteins.