The high affinity interleukin-6 (IL-6) signaling complex consists of IL- 6 and two membrane-associated receptor components: a low affinity but specific IL-6 receptor and the affinity converter/signal transducing protein gp130. Monomeric (IL-6(M)) and dimeric (IL-6(D)) forms of Escherichia coli-derived human IL-6 and the extracellular ('soluble') portions of the IL-6 receptor (sIL- 6R) and gp130 have been purified in order to investigate the effect of IL-6 dimerization on binding to the receptor complex. Although IL-6(D) has a higher binding affinity for immobilized sIL-6R, as determined by biosensor analysis employing surface plasmon resonance detection, IL-6(M) is more potent than IL- 6(D) in a STAT3 phosphorylation assay. The difference in potency is significantly less pronounced when measured in the murine 7TD1 hybridoma growth factor assay and the human hepatoma HepG2 bioassay due to time-dependent dissociation at 37°C of IL-6 dimers into active monomers. The increased binding affinity of IL-6(D) appears to be due to its ability to cross-link two sIL-6R molecules on the biosensor surface. Studies of the IL-6 ternary complex formation demonstrated that the reduced biological potency of IL-6(D) resulted from a decreased ability of the IL-6(D)-(sIL-6R)2 complex to couple with the soluble portion of gp130. These data imply that IL-6-induced dimerization of sIL-6R is not the driving force in promoting formation of the hexameric (IL- 6 · IL-6R · gp130)2 complex. A model is presented whereby the trimeric complex of IL-6R, gp130, and IL-6(M) forms before the functional hexamer. Due to its increased affinity for the IL-6R but its decreased ability to couple with gp130, we suggest that a stable IL-6 dimer may be an efficient IL-6 antagonist.