The objective of this study was to determine whether peptides consisting of the Ii-Key peptide LRMK linked to the N-terminal ends of HER-2 peptides would stimulate the expansion of antigen-specific E75-TCR+CD8+ cells. The peptides tested were N-acetylated and linked to an alpha-amide at the C-terminus; some of the peptides contained epsilon-aminovaleric acid (Ava) between the LRMK and the HER-2 peptide. Of the seven LRMK-HER-2 peptides tested to date, three effectively induced IFN-gamma production by peripheral blood mononuclear cells (PBMCs) from healthy donors and women with ductal carcinoma in situ. A fusion peptide, LRMK-Ava-HER-2(777-789), was more immunogenic than the natural HER-2(777-789) antigen, G89, with regard to IFN-gamma production. In combination with the CD8-activating peptide E75 [HER-2(369-377)] LRMK-p776 and LRMK-Ava-F7 induced the proliferation of E75-TCR(Med+Hi) CD8+ cells to a greater extent than did 1,000 or 5,000 nM of E75 alone, respectively. The induction effects were strongest at 600 nM for LRMK-p776 and 3,000 nM for LRMK-Ava-F7. At 3,000 nM, LRMK-p776 was cytotoxic to PBMCs. LRMK-p776 and F7 had a similar specificity and preferences for binding HLA-DR molecules. The molecular modeling of HLA-DR:LRMK-p776 and HLA-DR:LRMK-Ava-F7 complexes revealed the side chains of the peptides, which pointed towards the T-cell receptor. Differences in side chain orientation introduced by various N-terminal extensions of MHC class II-bound peptides should be important for directing CD4+ cells to stimulate CD8+ cells or for eliminating regulatory T cells in cancer immunotherapy.