The involvement of the CD43 molecule in the activation of mouse small intestinal intraepithelial lymphocytes (IELs) has been studied using a panel of twenty-two regulatory and effector immune response analytes. In the absence of stimulation in vitro, IELs produced low levels of CCL5 only. Upon CD3 stimulation, the activity of seven of twenty-two analytes was elevated relative to unstimulated cultures, including several proinflammatory cytokines and chemokines. Notably, CD3 stimulation in the presence of CD43 costimulation resulted in elevated levels of five analytes (interleukin-2, interferon-gamma, CCL5, granulocyte colony-stimulating factor, and granulocyte-monocyte colony-stimulating factor) above that produced by CD3 stimulation alone. That CD43 costimulation was responsible for elevated cytokine/chemokine activity was confirmed at the transcriptional level by real-time PCR for IFN-gamma and CCL5, and by ELISA for IFN-gamma. These findings open the way to a better understanding of the process by which T cells are activated in the intestinal epithelium.