In the present study, we examined the role of the recently identified glycosylphosphatidylinositol (GPI)-anchored cell surface molecule BY55, assigned as CD160, in TCR signaling. CD160 is expressed by most intestinal intraepithelial lymphocytes and by a minor subset of circulating lymphocytes including NK, TCRgammadelta and cytotoxic effector CD8bright+CD28- T lymphocytes. We report that CD160, which has a broad specificity for MHC class Ia and Ib molecules, behaves as a co-receptor upon T cell activation. Anti-CD160 mAb enhance the CD3-induced proliferation of freshly isolated CD160-enriched peripheral blood lymphocytes and CD160+ T cell clones. Further, the engagement of CD160 receptors on normal clonal T lymphocyte populations lacking CD4, CD8 and CD28 molecules by MHC class I molecules results in an increased CD3-induced cell proliferation. Further, we found that CD160 co-precipitates with the protein tyrosine kinase p56lck and tyrosine phosphorylated zeta chains upon TCR-CD3 cell activation. Thus, we demonstrate that CD160 provides co-stimulatory signals leading to the expansion of a minor subset of circulating lymphocytes including double-negative CD4/CD8 T lymphocytes and CD8bright+ cytotoxic effector T lymphocytes lacking CD28 expression.