Peripheral tolerance of T cells is necessary because thymic deletion is not complete, and tissue-specific Ags exist outside the thymus. We have reported that persistent Ag is required to maintain peripheral tolerance in vivo. We suspect that the TCR signaling pathway in in vivo tolerized cells is compromised due to continuous exposure to the Ags. In this study, the TCR signaling events in these cells were investigated using TCR transgenic mice (2C mice) whose T cells are predominantly Ld alloantigen-specific CD8 cells. The 2C mice were thymectomized and then rendered tolerant to Ld Ag by allogenic heart transplantation plus short-term treatment with rapamycin. We found that 1) the in vivo tolerized CD8 cells have compromised intracellular Ca2+ flux upon mitogen stimulation; and 2) their cellular tyrosine proteins fail to be phosphorylated properly upon TCR cross-linking. These results indicate that the signaling pathway in the in vivo tolerized CD8 cells is indeed defective. We also found that 1) the tolerized CD8 cells have no characteristic surface markers; and 2) the allograft is probably the place where the rejection response is initiated according to the appearance of an early activation marker of T cells on graft-infiltrating cells.