Pretransplant exposure to allogeneic lymphocytes can result in donor-specific unresponsiveness and prolonged allograft survival. Intracellular signaling events have been described in anergic T cell clones, but the biochemical events underlying in vivo induced unresponsiveness have not been studied in detail. We employed a TCR transgenic mouse, bearing the 2C TCR, providing adequate numbers of homogenous peripheral T cells to study biochemical aspects of T cell unresponsiveness in vivo. 2C mice exposed to semiallogeneic lymphocytes (H-2b x H-2d) experienced prolonged H-2d cardiac allograft survival, and cells from these mice did not proliferate or make IL-2 in response to alloantigen (H-2d). Importantly, there were marked differences in TCR-associated tyrosine phosphorylation activation patterns. The targets for the unresponsive state appear to be diminished Lck activation and absent ZAP-70 and LAT (linker for activation of T cells) phosphorylation. Our study demonstrates that Ag-induced tolerance in vivo is accompanied by altered early TCR-mediated signaling events.