We raised monoclonal antibodies against human macrophage migration inhibitory factor (MIF), and developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) method highly specific for human MIF. The ELISA system utilizes a solid phase monoclonal antibody as a capture antibody and a horseradish peroxidase-conjugated monoclonal antibody as a detector antibody. We used this ELISA method to evaluate the serum level of MIF in 240 healthy volunteers (140 males and 100 females). We found no significant difference in MIF concentration with respect to age. A significant difference was found with respect to sex, with the mean value (+/- SD) for male subjects of 5.3+/-2.3, and that for female subjects of 4.6+/-2.3 ng/ml (p<0.05). We next measured the serum MIF contents of patients with autoimmune diseases, and found that MIF levels were significantly elevated in patients with systemic lupus erythematosus and rheumatoid arthritis, 20.0+/-11.0 ng/ml and 21. 7+/-11.2 ng/ml, respectively. Using anti-MIF antibody-immobilized sepharose column chromatography, we discovered for the first time that MIF was present in erythrocytes. Taken together these results suggest that MIF plays a major role in autoimmune diseases and, moreover, potentially induces various patho-logical outcomes in cases of hemolytic disorders.