The homodimeric form of a recombinant cytokine interleukin-6 (IL-6(D)) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6(D) were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 (15)N-labeled and unlabeled ((14)N) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6(D) was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from cross-linked peptides of intermolecular origin are labeled with [(15)N/(15)N] + [(15)N/(14)N] + [(14)N/(15)N] + [(14)N/(14)N] yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with [(15)N] + [(14)N] yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated "mixed isotope cross-linking" MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6(D) having a domain-swapped fold similar to IL-10 and interferon-gamma. This fold implies that IL-6(D)-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6(D), rather than direct blocking of gp-130-binding sites on IL-6(D).