In this study we investigated recognition by ovarian tumor associated lymphocyte (OVTAL), and breast tumor associated lymphocytes (BRTAL), of peptides corresponding to the sequence 125-135 of the Aminoenhancer of split (AES) protein. Three of these peptides designated as G75:AES1/2 (128-135), G60: AES1/2 (127-137) and G61: AES1/2 (125-133) correspond to the wildtype AES sequence, while the fourth G76:GPLTPLPV, AES1/2 (128-135) corresponds to a variant sequence of the peptide G75 with the N-terminal Leu substituted to glycine. These sequences were chosen for study because mass-spectrometric analysis (MS) of a CTL active HPLC peptide fraction eluted from immunoaffinity precipitated HLA-A2 molecule, revealed: (a) the presence of an ion with a mass-to-charge ratio (m/z) of 793 which was more abundant than other ions of similar masses; (b) the tentatively reconstituted sequence of the ion 793 matched the sequence of peptide G76. We found that AES peptides G75 (128-135) and G76 (128-135) (L128G) reconstituted CTL recognition at concentrations ranging between 200-500 nM. These concentrations are lower than concentrations reported to activate effector function of CTL recognizing other epithelial tumor Ag. Furthermore, analysis with cloned CD8+ T cells indicated that G75 and G76 were not cross-reactive specificities, suggesting a key role for the N-terminal residues of the variant peptide in dictating specificities. Since the AES proteins are part of a set of transcriptional repressors encoded by the Enhancer of split [E(spl)] genes, and since these repressors are activated to suppress cell differentiation in response to Notch receptors signalling, the AES peptides may represent a novel class of self-antigens that deserve further consideration as tumor Ag in epithelial cancers.