The OVA 323–339 epitope recognized by DO11. 10 (H-2 d) and OT-II (H-2 b) T cells was investigated using amino-and carboxy-terminal truncations to locate the approximate ends of the epitopes and single amino acid substitutions of OVA 323–339 to identify critical TCR contact residues of the OVA 323–339 peptide. DO11. 10 and OT-II T cells are both specific for a C-terminal epitope whose core encompasses amino acids 329–337. Amino acid 333 was identified as the primary TCR contact residue for both cells, and amino acid 331 was found to be an important secondary TCR contact residue; however, the importance of other secondary TCR contact residues and peptide flanking residues differ between the cells. Additional OVA 323–339-specific clones were generated that recognized epitopes found in the N-terminal end or in the center of the peptide. These findings indicate that OVA 323–339 can be presented by IA d in at least three binding registers. This study highlights some of the complexities of peptide Ags such as OVA 323–339, which contain a nested set of overlapping T cell epitopes and MHC binding registers.