The activation of CD8(+) T cells by normal intestinal epithelial cells in antigen-specific or allogeneic mixed cell culture systems has significant implications for the modulation of mucosal immune responses due to the fact that these T cells appear to have regulatory rather than cytolytic activity. A 180-kDa glycoprotein (gp180) has been identified and shown to be important in CD8(+) T cell activation by intestinal epithelial cells. In this study, we examine, in further detail, the role that the CD8 molecule plays in this interaction. It has been previously shown that monoclonal antibodies against gp180 inhibited the activation of CD8-associated p56(lck) in T cells. Although indirectly suggested by these data, there was no evidence that the activation of this protein tyrosine kinase was a direct result of gp180 interacting with the CD8 molecule. In this study, we document that soluble gp180 is able to bind to CD8-Fc fusion proteins and is absorbed by human CD8 alpha but not CD4 transfected murine T cells and that this interaction is dependent upon carbohydrate on the gp180 molecule. Furthermore, the sites used for binding by gp180 are distinct from those used by the conventional CD8 ligand, class I MHC. Thus, gp180 appears to be a novel CD8 ligand that plays an important role in the activation of CD8-associated kinases and of CD8(+) T cells.