Clonal anergy as a mechanism for tolerance in T lymphocytes can be studied using an in vitro culture system, in which cloned CD4+ Th1-type murine T cells are rendered anergic for IL-2 transcription. The long-lasting molecular changes in anergic cells that prevent the response to Ag restimulation are not yet known. To determine whether the TCR might be uncoupled from normal intracellular signaling pathways, we investigated the response of anergic T cells to Ag, to anti-CD3 antibodies, or to anti-CD4 antibody restimulation in terms of early protein tyrosine phosphorylation events. Tyrosine phosphorylation of the CD3 zeta chain was apparently normal. In contrast, defects in the induction of tyrosine phosphorylation of three major T cell protein substrates were demonstrated. Altered phosphorylation correlated with functional nonresponsiveness for proliferation and reversal of anergy by growth in exogenous IL-2 resulted in reversal of the phosphorylation defects as well as in recovery of Ag responsiveness. These results suggest that specific defects in tyrosine phosphorylation pathways required for the induction of IL-2 synthesis may help to explain nonresponsiveness to Ag in tolerant T cells.