Biochemical and genetic investigations of thymocyte negative selection will be facilitated by the availability of cultured cell lines representing immature thymocytes, but to date thymocyte lines have demonstrated resistance to Ag receptor-mediated death. It is shown here that a CD4+CD8+ TCR alpha beta+ cell line with a TCR of known Ag and class II MHC specificity (cytochrome c with I-Ek) undergoes Ag dose-dependent apoptotic death in a manner consistent with negative selection of normal thymocytes. In contrast to superantigen-induced selection, apoptosis in this model is unaffected by blocking co-engagement of CD4 and the TCR. At high Ag doses, a significant fraction of thymocytes becomes committed to apoptosis within 2 h of contact with antigen-pulsed APCs, while increased interaction times increase the probability of death. Longer contact periods are required at lower Ag doses. These kinetics of commitment to apoptosis suggest that, in normal negative selection, a thymocyte clone may be deleted after any of several encounters with MHC-bearing cells during progress through the thymus. They also suggest why negative selection predominates when a specific peptide is competent both to delete and to select positively.