Migration-inhibitory factor (MIF) is a lymphokine that acts to localize mononuclear phagocytes (monocytes and macrophages) and perhaps to activate them. Mo cells are a human T cell leukemia virus II-infected T cell line previously shown to secrete large quantities of MIF upon stimulation with phytohemagglutinin and phorbol myristate acetate. MIF was purified from Mo cell-conditioned medium by gel filtration, phenyl- Sepharose affinity chromatography, isoelectrofocusing, and reverse- phase high-performance liquid chromatography (RP-HPLC). Overall purification was 6,000-fold. The purified MIF fraction was found to display potent colony-stimulating factor (CSF) activity when assayed on human bone marrow cells. The double peak of MIF activity as shown by C 18-RP-HPLC coincided with the double peak of CSF activity. A monoclonal antibody selected for its anti-MIF activity absorbed both the CSF and the MIF activity. These findings indicate that MIF and CSF are either identical molecules or closely related molecules with common structural elements.