In this study, the phenotype, TCR signaling events, and function of T cells developed de novo during adulthood in the presence of extrathymic alloantigen were investigated. C57BL/6 mice(H-2b) were first transplanted heterotopically with BALB/c hearts (H-2d) and treated with rapamycin for 2 wk to create a tolerant status. Three weeks postoperation, the mice were whole body irradiated and transplanted with bone marrow cells from 2C mice, which are transgenic for TCR, and most of their T cells are Ld-specific CD8 cells. The 2C T cells developed de novo in the C57BL/6 mice were not able to reject the heart allograft. No clonal deletion, TCR down-regulation, or CD8 down-regulation was found in the tolerized 2C T cells. There was no characteristic phenotype of these cells in terms of CD25, ICAM-1, CD44, and MEL-14 expression. Early TCR signaling events such as intracellular calcium concentration flux, tyrosine phosphorylation, Lck and Fyn kinase activities, and Lck and Fyn protein levels in the tolerized 2C T cells were comparable to their normal counterparts, but the tolerized T cells were defective in IL-2 production and proliferation upon H-2d alloantigen stimulation in vitro. Exogenous IL-2 could not reverse the compromised proliferation. The results of this study indicate that during adulthood, the de novo-developed T cells become tolerant to extrathymic Ag without clonal deletion. These newly minted T cells are functionally defective although they are indistinguishable from normal T cells in phenotypes and in some early signaling events.