Gas phase, time-resolved, thermal dissociation measurements are reported for three protein-ligand complexes, the homodimer alpha(1 -> 3)galactosyltransferase (GTB) with its native disaccharide acceptor, alpha-L-Fucp-(1 -> 2)-beta-d-Galp, the homotetramer streptavidin and its specific ligand biotin, and the homodimer of wheat germ agglutinin (WGA) and the disaccharide beta-D-GlcNAcp-(1 -> 6)-D-Galp. It is shown that protein-ligand interactions that are structurally and thermodynamically equivalent in solution exhibit identical kinetic and energetic stabilities in the gas phase. In contrast, nonequivalent interactions in solution give rise to kinetically nonequivalent interactions in the gas phase. The equivalency of the rate constants measured for the loss of ligand from multiple, equivalent binding sites within a given protein complex is compelling evidence that the binding sites are structurally equivalent in the gas phase. This is the first quantitative experimental evidence for the preservation of elements of structural equivalency within protein complexes upon transfer from solution to the gas phase. This important finding serves as the basis for a new assay, which employs time-resolved tandem mass spectrometry, for evaluating the equivalency of ligand binding sites within proteins and multiprotein complexes in solution.