In recent years, synthetic peptides have been utilized extensively to characterize the minimal essential immunodominant sites on model protein Ag. However, little work has focused on the effect that sequences flanking these minimal recognition sites may exert on T cell recognition. Previous work with staphylococcal nuclease (Nase) demonstrated that I-Ek-restricted clones recognize the peptide 81-100, whereas I-Ab-restricted clones recognize the over-lapping but non-cross-reacting peptide 91-110. Further analysis with 15 or 10 residue peptides within the region 81-110 reveals that the minimal sequence capable of stimulating I-Ek-restricted clones is contained within the decapeptide 91-100. Addition of residues 86-90, to give the peptide 86-100, enhanced the recognition substantially, whereas addition of residues 101-105 produced a 91-105 peptide with no stimulatory ability. These results suggest that interactions between the antigenic peptide 91-100 and residues within the flanking 101-105 sequence have negative consequences for presentation of the immunodominant epitope to T cell clones. Introduction of single amino acid substitutions within 91-105 produced peptides that induce responses comparable to those seen with 91-100. These results are consistent with the suggestion of negative interactions between the minimal immunodominant site and flanking sequences in that single residue substitutions may remove these negative interactions and lead to restoration of stimulatory ability. The negative effect of flanking sequences on T cell recognition of immunodominant sites presents new considerations for development of synthetic vaccines as well as for understanding the biology of Ag processing and presentation.