Generation and adoptive transfusion of alloreactive cytotoxic T lymphocytes (CTLs) specific for tumor are expected to circumvent tumor tolerance. Here we describe a novel protocol to raise allo-restricted, peptide-specific CTLs by co-culture of murine splenocytes and autologous macrophage bearing an allogeneic H-2K molecule associated with its restricted peptide (peptide/allo-MHC). The extracellular domains of H-2K(d) were fused with constant domains of murine IgG2a heavy chain to generate a fusion protein (peptide/H-2K(d)/IgG2aFc, the dimer) consisting of divalent TCR-ligands and an IgG Fc receptor type I (FcgammaRI)-reactive moiety. The dimer is able to bind to macrophage (Mvarphi) of H-2K(k) via the interaction of the Fc part with FcgammaRI, and cause the H-2K(k) Mvarphi to be coated with the peptide/H-2K(d) complex. The results show that proliferation of CD8+ cells is enhanced and that the specific-tetramer stained CD8+ cells appear more frequently by co-culture of H-2K(k) splenocytes with the autologous Mvarphi loaded with the dimer. Furthermore, the CD8+ T cells from the co-cultural bulk exhibit an elevated cytotoxicity against a specific target (H-2K(d)-restricted, peptide-specific cytotoxicity), compared with that against the irrelevant targets. This study provides a strategy for preparation of allo-restricted, peptide-specific CTLs, which may add to our arsenal for adoptive immunotherapy to eliminate chronic virally infected or tumor cells.