The availability of T-cell mutants that lack one or more of the T-cell antigen receptor chains, and clones of the genes that encode these chains, has allowed us to reconstruct a cell surface TCR that expresses structurally mutated zeta chains. By sequentially removing each of the intracytoplasmic tyrosine residues, it has been possible to determine that zeta phosphorylation is a cooperative process, with each of the 4 carboxyl tyrosines contributing to the phosphorylation pattern after activation via the TCR. Furthermore, under no circumstances did we find that tyrosine phosphorylation had any effect upon activation-induced IL- 2 secretion. Thus, a functional role for zeta phosphorylation on tyrosine residues remains to be defined. Heterochimeric antibodies (made of 2 Fab fragments, one against the TCR and the other against an MHC class II molecule) were used to stimulate T cells with mutated TCR zeta chains. When bound to plastic, the antibodies stimulated these mutants and the wild type equally, but when "presented" by an accessory cell only the wild type responded. In contrast to immobilized antibody, plastic-adherent antigen/MHC class II was able to stimulate only the wild type cell. The data strongly support a novel model holding that stable aggregation of TCRs is essential for signaling to proceed, and that a full length zeta chain is necessary for occupancy with Ag/MHC to induce such an aggregate, perhaps by physically interacting with the other components of the signaling pathway or the cytoskeleton.