The current study examines how responsiveness of T cells is affected by the avidity of the peptide/MHC engaged during positive selection of their thymocyte precursors. We used a thymus reaggregate culture system in which CD4(+)CD8(+) thymocytes from AND TCR transgenic mice were induced to undergo positive selection by pigeon cytochrome c (PCC) peptide or its analogs presented by I-E(k) class II MHC on a thymic epithelial cell line. When low-affinity peptide analogs drove positive selection, up to 100 microM was needed to produce >50% CD4(+) T cells, and these cells were highly responsive to PCC. In contrast, <0.2 microM high-affinity peptides was required to achieve similar selection efficiency, but the resultant cells failed to respond to PCC. However, these cells were not dead based on dye exclusion and capacity to respond to phorbal ester and to agonist if IL-2 was also present, supporting the view that non-responsiveness of cells selected on high-affinity peptides is a form of central T cell tolerance distinct from deletion. Cells selected on intermediate-affinity peptides showed variable responsiveness which was suppressed 5- to 10-fold by addition during reaggregate culture of antibody to the IL-7R. Similarly, supplementary IL-7 in the reaggregate culture produced CD4(+) T cells that were promiscuously responsive. Overall, this study demonstrates that the responsiveness of T cells is not rigidly controlled and that the presence of IL-7 during T cell development has the potential to negate central T cell tolerance and produce autoreactive T cells.