Development of immature CD4⁺CD8⁺ thymocytes into functionally mature CD4⁺ and CD8⁺ T cells is driven by selection events that require signals transduced through the T cell antigen receptor (TCR). Transduction of TCR signals in the thymus involves tyrosine phosphorylation of the protein tyrosine kinase ZAP-70 by p56^lck and results in induction of ZAP-70 enzymatic activity. We have identified a novel, spontaneously arising point mutation within a highly conserved motif (DLAARN) in the kinase domain of murine ZAP-70 that uncouples tyrosine phosphorylation of ZAP-70 from induction of ZAP-70 kinase activity. Mice homozygous for this mutation are devoid of mature T cells because thymocyte development is arrested at the CD4⁺CD8⁺ stage of differentiation. The developmental arrest is due to the inability of CD4⁺CD8⁺ thymocytes to propagate TCR signals in the absence of ZAP-70 kinase activity despite tyrosine phosphorylation of TCR-associated ZAP-70 molecules.