Recombinant immunoreceptors with specificity for the carcinoembryonic Ag (CEA) can redirect grafted T cells to a MHC/Ag-independent antitumor response. To analyze receptor-mediated cellular activation in the context of CD28 costimulation, we generated: 1) CEA+ colorectal tumor cells that express simultaneously B7-1 and B7-2, and 2) CEA-specific immunoreceptors that harbor intracellularly the signaling moities either of CD28 (BW431/26-scFv-Fc-CD28), CD3ζ (BW431/26-scFv-Fc-CD3ζ), or FcεRIγ (BW431/26-scFv-Fc-γ). By retroviral gene transfer, we grafted activated T cells from the peripheral blood with these immunoreceptors. T cells that express the FcεRIγ or CD3ζ signaling receptor lysed specifically CEA+ tumor cells and secreted high amounts of IFN-γ upon receptor cross-linking, whereas anti-CEA-CD28 receptor-grafted T cells did not, indicating that CD28 signaling alone is not sufficient for efficient T cell activation. CD28 costimulation did not affect cytolysis by T cells equipped with γ-or ζ-signaling receptors, but enhanced both IFN-γ secretion and proliferation. CD28 costimulation, however, was required for efficient IL-2 secretion of anti-CEA-γ receptor-grafted T cells. Both purified CD4+ and CD8+ T cells grafted with immunoreceptors required CD28 costimulation for complete T cell activation. We integrated both CD28 and CD3ζ signaling domains into one combined immunoreceptor molecule (BW431/26-scFv-Fc-CD28/CD3ζ) with dual signaling properties. T cells grafted with the combined CD28/CD3ζ signaling receptor secreted high amounts of IL-2 upon Ag binding without exogenous B7/CD28 costimulation, demonstrating that both MHC-independent cellular activation and CD28 costimulation for complete T cell activation can be delivered by one recombinant receptor molecule.