CD8(+) regulatory T cells (Tregs) play an important role in regulating peripheral immune tolerance. However, difficulties in the characterization of CD8(+) Tregs that lack suitable markers have a considerably limited research in this area. Moreover, the induction and effector mechanisms of CD8(+) Tregs remain unclear. Herein, we demonstrate the suitability of Ly49 and CD44 as markers for CD8(+) Tregs. Our data also show that MHC class II restricted peptides induce CD8(+)CD44(+)Ly49(+) Tregs via CD4(+) T cell activation. Furthermore, we also found cross-suppressive activity of these CD8(+) Tregs on responding CD4(+) T cells in a cytotoxicity dependent manner. Our data provide new insights into the induction and function of CD8(+) Tregs.