CD8⁺CD122⁺ regulatory T cells are a newly identified, naturally occurring type of regulatory T cell that produce interleukin‐10 (IL‐10) and effectively suppress interferon‐γ (IFN‐γ) production from both CD8⁺ and CD4⁺ target cells. Molecular mechanisms responsible for the recognition of target cells by CD8⁺CD122⁺ regulatory T cells were investigated in this study by using an in vitro culture system that reconstitutes the regulatory action of these cells. CD8⁺CD122⁺ regulatory T cells did not produce IL‐10 and did not suppress the IFN‐γ production of allogeneic target T cells when they were stimulated by immobilized anti‐CD3 antibody alone, but they clearly produced IL‐10 and suppressed the IFN‐γ production of target cells when stimulated by anti‐CD3 plus anti‐CD28‐coated beads. IFN‐γ production by major histocompatibility complex‐class I‐deficient T cells was also suppressed by CD8⁺CD122⁺ regulatory T cells stimulated with anti‐CD3 plus anti‐CD28 antibody but was not suppressed by cells stimulated by anti‐CD3 alone. Experiments examining the blockade of cell surface molecules expressed on either the regulatory cells or the target cells by adding specific neutralizing antibodies in the culture indicated that CD80, CD86, and CD28 molecules were involved in the regulatory action, but cytotoxic T lymphocyte antigen‐4, inducible costimulatory molecule (ICOS) and programmed death‐1 (PD‐1) molecules were not. Finally, CD8⁺CD122⁺ cells isolated from CD28‐knockout (CD28^−/−) mice showed no regulatory activity. These results indicate that CD8⁺CD122⁺ regulatory T cells recognize target T cells via the interaction of CD80/CD86–CD28 molecules to become active regulatory cells that produce suppressive factors such as IL‐10.