We recently showed that oligomerization of CD40 molecules on cell surface leads to disulfide-linked CD40/CD40 dimer formation, an event that is necessary for CD40-induced B7-2 expression in human B cells. Here, we demonstrate that CD40/CD40 dimers formation also occurs in different cell types such as T24 bladder cancer cells and CD40-transfected HEK 293 cells. Disulfide bonds mediate the formation of CD40/CD40 homodimers in CD40-activated cells. To determine the potential residue(s) involved in disulfide bonds formation and subsequent CD40-induced IL-8 expression, we generated a CD40 mutant in which the extracellular cysteine 6 was replaced by a glutamine (CD40-C6Q). CD40-induced IL-8 mRNA expression and protein synthesis were studied in stably transfected HEK 293 cells that were sorted out along with similar levels of expression of wild type (CD40-WT) and CD40-C6Q molecules. In contrast to cells expressing CD40-WT protein, disulfide-linked CD40/CD40 dimer formation was completely abolished in HEK 293 cells expressing CD40-C6Q proteins. Abolishment of disulfide-linked CD40/CD40 dimers in these transfected cells was sufficient to inhibit CD40-induced mRNA expression and secretion of IL-8. This study identifies the extracellular cysteine 6 of CD40 molecules as a potential molecular target to disrupt the expression of CD40-induced pro-inflammatory cytokines by epithelial cells.