The structure of the cytokine MIF has been investigated by X-ray crystallography, NMR, and biochemical methods with conflicting results regarding the structural and functional oligomerization state of this protein. Determination of the oligomeric state(s) is important for understanding more precisely the molecular mechanism of MIF action. To address this issue, we performed cross-linking of human and mouse MIF and selected mutants by various methods and analyzed the oligomerization by SDS-PAGE and gel filtration. MIF was found to form a mixture of monomeric, dimeric, and trimeric states at physiological concentrations, with the monomer and dimer representing the major species. Similar results were obtained when the carboxy-truncated mutants MIF(1-104) and MIF(1-109) were examined, indicating that the C-terminus of MIF is not critical for trimer stabilization. Cross-linking analysis of the isosteric Cys --> Ser mutants C56S and C80S of human MIF resulted in a similar oligomer distribution, whereas substitution of Cys59 led to a significant reduction in the dimeric and trimeric forms, indicating that the hydrophobic region around Cys59 is important for the oligomerization of MIF. Together, our data argue that physiological MIF solutions contain a mixture of monomers, dimers, and trimers.