Previous studies have shown that normal intestinal epithelial cells (IECs) are able to selectively activate CD8(+) T cells with suppressor activity, inducing proliferation associated with the activation of both the CD8-associated kinase p56(lck) and the T cell receptor (TCR)-associated kinase p59(fyn). This process appears to relate in part to a 180-kDa IEC surface glycoprotein, gp180, which binds to CD8 and activates CD8-associated p56(lck). However, purified gp180 alone is unable to induce T cell proliferation and does not activate p59(fyn). Because the class Ib molecule CD1d is expressed by IECs and monoclonal antibodies (mAbs) against CD1d inhibit IEC-induced proliferation of CD8(+) T cells, co-immunoprecipitation and enzyme-linked immunosorbent assay studies were performed, which demonstrated an association of gp180 and CD1d on the IEC surface. Interestingly, the activation of p59(fyn) in IEC-T cell co-cultures was blocked by the anti-CD1d mAb D5 but not by the anti-gp180 mAb B9. Conversely, treatment of IECs with mAb B9 inhibited IEC-induced activation of p56(lck) but not p59(fyn). More directly, a human CD1d cDNA (FO-1 D5) transfectant was able to activate p59(fyn) but not p56(lck). These data suggest that the CD1d-gp180 complex on the surface of IECs can be recognized by the TCR-CD8 co-receptor, resulting in the activation of CD8(+) T cells.