To examine the effects of nuclear factor kappa B (NF-kappaB) inhibition on the secretion of macrophage migration inhibitory factor (MIF) in human CD4(+) T cells. Isolated human CD4(+) T cells were cultured for 24h with pharmacological inhibitors of NF-kappaB including parthenolide, pyrrolidine dithiocarbamate, BAY 11-7082, gliotoxin, oridonin, andrographolide, and NF-kappaB shRNA. MIF concentration was measured by intracellular flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. The intracellular concentrations O(2)(-), H(2)O(2), and glutathione were measured using the oxidation-sensitive fluorescent dyes dihydroethidium, dichlorodihydrofluorescein diacetate, and monochlorobimane, respectively. The amount of phosphorylated c-Jun was measured by Western blotting. Treatment of CD4(+) T cells with NF-kappaB inhibitors significantly increased MIF concentration in culture supernatants, MIF gene expression, and O(2)(-) production, and decreased the intracellular concentrations of MIF, H(2)O(2), and glutathione. Treatment with LY294002 (PI3K inhibitor) and SP600125 (JNK inhibitor) suppressed NF-kappaB inhibitor induced MIF mRNA expression and MIF secretion. LY294002 and SP600125 inhibited the parthenolide-induced phosphorylation of c-Jun. Treatment with H(2)O(2) decreased the amount of intracellular MIF protein and increased MIF concentration in the culture supernatant. N-acetylcysteine, an antioxidant precursor of glutathione, inhibited the parthenolide-induced and H(2)O(2)-induced secretion of MIF. These results indicate that pharmacological inhibition of NF-kappaB causes the release of MIF through de novo synthesis of MIF and the secretion of preformed MIF in CD4(+) T cells through the production of reactive oxygen species.