Macrophage migration inhibitory factor (MIF) is known to exert pleiotropic functions including inhibition of macrophage migration, anchoring, and counteraction of the anti-inflammatory and immunosuppressive activity of glucocorticoids. Ninety-three primary breast cancer tissues and 64 sera of primary breast cancer patients were analyzed for the expression of MIF. The clinico-pathological significance of MIF expression was evaluated. It was found that MIF was frequently over-expressed in primary breast cancer tissues. RT-PCR and western blotting analysis confirmed that wild-type MIF is expressed, and immunohistochemical analysis showed that MIF expression was localized at tumor cells as well as stromal cells, including tumor-associated macrophages. Intratumoral MIF protein concentrations detected by enzyme-linked immunosorbent assay (ELISA) varied with a median value of 1821 ng/mg protein (range: 8 - 8126 ng/mg protein), and correlated inversely with nodal involvement (P = 0.039). No significant correlation was observed with other clinico-pathological factors including tumor size, menopausal status and hormone receptors. The circulating level of MIF protein ranged up to 105.7 ng/ml (median: 17.3 ng/ml), and it was also found to correlate inversely with the number of involved nodes (P = 0.02). A comparative study with other soluble inflammatory mediators showed that intratumoral levels of MIF were significantly associated with those of interleukin-1 beta, suggesting that interactions between tumor cells and tumor-associated macrophages play an important role in the up-regulation of MIF. The multifunctional inflammatory/immune mediator MIF was frequently expressed in primary breast cancer, and its expression level was inversely associated with nodal spread. Thus, MIF seems to play a role in tumor-stroma interactions of primary breast cancers, particularly those with a phenotype of node-negative or minimal nodal spread.