BACKGROUND & AIMS: Immune responses to innocuous intestinal antigens appear tightly controlled by regulatory T lymphocytes. While CD4+ T lymphocytes have recently attracted the most attention, CD8+ regulatory T-cell populations are also believed to play an important role in control of mucosal immunity. However, CD8+ regulatory T-cell function has mainly been studied in vitro and no direct in vivo evidence exists that they can control mucosal immune responses. We investigated the capacity of CD8+CD28- T cells to prevent experimental inflammatory bowel disease (IBD) in mice. METHODS: CD8+CD28- regulatory T cells were isolated from unmanipulated mice and tested for their capacity to inhibit T-cell activation in allogeneic mixed lymphocyte cultures in vitro and to prevent IBD induced by injection of CD4+CD45RB(high) cells into syngeneic immunodeficient RAG-2 mutant mice. RESULTS: CD8+CD28- T lymphocytes inhibited proliferation and interferon gamma production by CD4+ responder T cells in vitro. CD8+CD28- regulatory T cells freshly isolated from spleen or gut efficiently prevented IBD induced by transfer of colitogenic T cells into immunodeficient hosts. Regulatory CD8+CD28- T cells incapable of producing interleukin-10 did not prevent colitis. Moreover, IBD induced with colitogenic T cells incapable of responding to transforming growth factor beta could not be prevented with CD8+CD28- regulatory T cells. CD8+CD28+ T cells did not inhibit in vitro or in vivo immune responses. CONCLUSIONS: Our findings show that naturally occurring CD8+CD28- regulatory T lymphocytes can prevent experimental IBD in mice and suggest that these cells may play an important role in control of mucosal immunity.