We set out to determine whether thymocytes from T cell receptor (TCR) transgenic animals specific for a class I‐restricted determinant from ovalbumin (OVA) showed the same fine specificity for antigen‐driven deletion in single‐cell suspension culture as required for mature T cell activation. The transgenic TCR is specific for the K^b‐restricted peptide OVA_257‐264 (SIINFEKL) which is known to have four TCR contact residues at position 1, 4, 6, and 7 from the crystal structure of this fragment in complex with K^b. OVA_257‐264 analogs systematically substituted at each of these positions were assayed for their ability to promote immature double‐positive thymocyte deletion or mature T cell activation of a cytotoxic T lymphocyte line derived from this transgenic mouse. In the absence of additional antigen‐presenting cells, single‐cell thymocyte suspensions showed that the specificity for double‐positive thymocyte deletion and mature T cell activation was virtually identical, demonstrating a limited cross‐reactivity with a number of variants having conservative substitutions at these exposed residues. These peptides were considerably more efficient at both thymic deletion and mature T cell activation than a number of non‐conservative substitution analogs known to act as antagonists of OVA_257‐264 and capable of selecting transgenic T cells in thymic organ culture. Therefore, both peripheral T cell activation and thymic deletion have an overall similar pattern of peptide specificity which differs from that required for positive selection. This suggests that a subset of major histocompatibility complex‐presented peptides could promote positive selection without causing either thymic deletion or peripheral activation of those selected T cells.