The potential role in cell growth of Macrophage migration inhibitory factor (MIF) has been studied, however, the mechanism of its anti-tumor effect is poorly understood. Antisense-MIF plasmids were directly injected into colon 26 tumors embedded in the back of mice. Furthermore, the role of MIF in the cell cycle was assessed with regard to retinoblastoma (Rb) protein and transcription factor E2F. Plasmids containing sense- and antisense-MIF genes were transfected into human colon cancer KM12SM cells in vitro. To examine the Rb protein-E2F pathway, plasmids containing each specific cis-acting enhancer for Rb protein and E2F with luciferase reporter genes, pRB-luc and pE2F-luc, respectively, were used. Antisense MIF treatment significantly reduced the tumor size. In vitro cell proliferation was significantly suppressed by the antisense treatment as examined by BrdU uptake. Transcriptions of Rb protein were 8.4x10(3) (RLU), 9.5x10(3) and 24.3x10(3) in the antisense MIF, PBK, and the sense MIF, respectively. As for E2F, transcription activities were 3.8x10(3), 3.6x10(3) and 7.7x10(3), respectively. These results indicate the possibility that MIF may promote tumor growth, in which the activation-inactivation mechanism of the Rb protein-E2F pathway could be profoundly involved.