One of the strategies to improve the outcome of anti-erbB2-mediated immunotherapy is to combine anti-erbB2 antibodies with T-cell-based adoptive immunotherapy, which can be achieved by expressing anti-erbB2 mAb on the surface of T cells. A single-chain variable fragment (scFv) from an anti-erbB2 mAb has been expressed on T cell surface to bind to erbB2-positive cells, and CD3 zeta has been expressed as a fusion partner at C terminus of this scFv to transduce signals. T cells grafted with this chimeric scFv/CD3 zeta were able to specifically attack target tumor cells with no MHC/Ag restriction. To test the effects of CD28 signal on cellular activation and antitumor effectiveness of chimeric scFv/CD3 zeta-modified T cells, we constructed a recombinant anti-erbB2 scFv/Fc/CD28/CD3 zeta gene in a retroviral vector. T cells expressing anti-erbB2 scFv/Fc/CD28/CD3 zeta specifically lyzed erbB2-positive target tumor cells and secreted not only interferon-gamma (IFN-gamma) but also IL-2 after binding to their target cells. Our data indicate that CD3 and CD28 signaling can be delivered in one molecule, which is sufficient for complete T cell activation without exogenous B7/CD28 co-stimulation.