Abstract We have expressed the human macrophage migration inhibitory factor (MIF) in Escherichia coliusing the pKP 1500 expression plasmid, which contains the tac promoter and a temperature-sensitive origin of replication, to ensure a high plasmid copy number at elevated temperatures. The recombinant protein accumulated intracellularly in soluble form. We have designed a simple two-step procedure for protein purification by gel filtration on Sephadex G-50 and cation exchange chromatography on CM cellulose columns. This results in significantly improved yields. One gram of recombinant human MIF was isolated from 50 g of E. colicells (wet weight). The 12.5-kDa protein was shown to be pure by SDS–PAGE, IEF, and HPLC. The identity of the purified protein was verified by N-terminal amino acid sequencing. The purified protein exhibits MIF activity. The near-UV CD and the 1H NMR spectra confirmed its highly ordered, native-like structure. The far-UV CD spectrum revealed that recombinant human MIF contains well-defined secondary structure.