Tumor necrosis factor-alpha (TNFalpha)-blocking therapy, using biologic TNFalpha antagonists, has been approved for the treatment of several diseases including rheumatoid arthritis, psoriasis and Crohn's disease. There have been few detailed studies of binding characterizations for the complex formation by TNFalpha and clinically relevant antagonists, particularly Infliximab (Remicade) and Etanercept (Enbrel). Here we characterized the binding stoichiometry and size of soluble TNFalpha-antagonist complexes and identified energetically important binding sites on TNFalpha for the three antagonists, Etanercept, Infliximab, and the recently developed humanized TNFalpha neutralizing monoclonal antibody, YHB1411-2. Size-exclusion chromatography and dynamic light scattering analyses revealed that the three antagonists formed distinct thermodynamically stable TNFalpha-antagonist complexes that exhibited differences in their size and composition. Energetically important binding residues on TNFalpha were identified for each antagonist by a sequence of experiments that consisted of competition binding assays, fragmentations, loop mutations, and single-point mutations using yeast surface-displayed TNFalpha, which was further confirmed for solubly purified TNFalpha mutants by surface plasmon resonance technique. Analyses of the binding geometry based on binding site location, spatial constraints, and valency satisfaction allowed us to interpret the thermodynamically stable complexes as follows: one molecule of Etanercept and one molecule of trimeric TNFalpha (Etanercept1-TNFalpha1), Infliximab6-TNFalpha3, and YHB1411-2(4)-TNFalpha2. The distinct features of the soluble antagonist-TNFalpha complex formation among the antagonists may give further insights into their different neutralizing mechanisms and pharmacokinetic profiles.