Macrophage migration inhibitory factor (MIF) has been shown to play an important role in macrophage-mediated diseases. We investigate the potential role of MIF in atherogenesis using a hypercholesterolemic rabbit model. New Zealand White rabbits fed with a 2% cholesterol diet developed hypercholesterolemia and early fatty streaks at 1 month. The lesions became advanced at 3 months and were associated with de novo MIF expression by vascular endothelial cells (VECs) and smooth muscle cells (SMCs), as demonstrated by immunohistochemistry, reverse transcriptase-polymerase chain reaction, and in situ hybridization. By contrast, there was no increase in MIF levels in rabbits fed a normal diet. In early atherogenesis, marked upregulation of MIF mRNA and protein by VECs and some intimal cells were closely associated with CD68(+) monocyte adhesion onto and subsequent migration into subendothelial space. Of significance, the accumulation of macrophages was exclusively localized to areas of strong MIF expression, which may be associated with the macrophage-rich fatty streak lesion formation. Upregulation of MIF by SMCs is transient during atherogenesis. Importantly, strong MIF expression by activated macrophages may be responsible for the development of foam cell-rich lesions. Finally, the ability of MIF to induce intercellular adhesion molecule-1 expression by VECs implicates its pathogenic role in atherogenesis. In conclusion, the present study provides the first demonstration that MIF is markedly upregulated during atherogenesis. Upregulation of MIF by VECs and SMCs may play a role in macrophage adhesion, transendothelial migration, accumulation, and, importantly, transformation into foam cells. Furthermore, strong MIF expression by macrophages may both initiate and amplify the atherogenesis process.