Macrophage migration inhibitory factor (MIF) is a cytokine that is involved in the regulation of inflammation as well as cell proliferation and differentiation. Deactivation of MIF by antibodies or inhibition of MIF binding to its receptor, CD74, attenuates tumor growth and angiogenesis. To discover small-molecule inhibitors of MIF's biological activity, virtual screening was performed by docking 2.1 million compounds into the MIF tautomerase active site. After visual inspection of 1200 top-ranked MIF-ligand complexes, 26 possible inhibitors were selected and purchased and 23 of them were assayed. The in vitro binding assay for MIF with CD74 revealed that 11 of the compounds have inhibitory activity in the micromolar regime, including four compounds with IC(50) values below 5 microM. Inhibition of MIF tautomerase activity was also established for many of the compounds with IC(50) values as low as 0.5 microM; Michaelis-Menten analysis was performed for two cases and confirmed the competitive inhibition.