T cells of tumor bearers often show defective TCR-mediated signaling events and, therefore, exhibit impaired immune responses. As such, patients with heavy tumor burden are often not amenable to adoptive T cell therapy. To overcome this limitation, we have developed a chimeric receptor that joins an extracellular single chain Fv (scFv) of a specific Ab for Ag recognition to an intracellular protein tyrosine kinase (PTK) for signal propagation. Stimulation through the scFv-PTK receptor should bypass defective TCR-proximal events and directly access the T cell's effector mechanisms. In this study we describe the optimization of a scFv-PTK configuration, leading to complete T cell activation. The cytosolic PTK Syk is superior to its family member, Zap-70, for intracellular signaling. As a transmembrane (TM) domain, CD4 performs better than CD8 when plastic-immobilized Ag serves as a stimulator. However, when APC are used to trigger chimeric receptors, the need for a flexible spacer between the scFv and TM domains becomes apparent. The CD8alpha-derived hinge successfully performs this task in chimeric scFv-Syk receptors regardless of its cysteine content. A cytotoxic T cell hybridoma expressing chimeric receptor genes composed of scFv-CD8(hinge)-CD8(TM)-Syk or scFv-CD8(hinge)-CD4(TM)-Syk is efficiently stimulated to produce IL-2 upon interaction with APC and specifically lyses appropriate target cells in a non-MHC-restricted manner.