AbstractIn recombinase-deficient (RAG-2−/−) mice, double-negative thymocytes can be stimulated to proliferate and differentiate by anti-CD3 Abs. CD3 molecules are expressed on the surface of these cells in association with calnexin. In this study, we show that ζ-chains can be recovered as phosphorylated proteins in association with phosphorylated ZAP-70 from anti-CD3-stimulated RAG-2−/− thymocytes, even though they are not demonstrably associated with the CD3/calnexin complex. The lack of a physical association of ζ dimers with the CD3 complex in RAG-2−/− thymocytes and also in a pre-TCR-expressing cell line, as well as the efficient association of ζ dimers with ZAP-70 in the RAG-2−/− thymocytes, suggest that these ζ-chain dimers could contribute to pre-TCR signaling. This idea is supported by the finding that in RAG-2−/− ζ-deficient thymocytes, ZAP-70 and p120cbl were only weakly phosphorylated.