Interleukin-27 (IL-27) is a new IL-12-related heterodimeric cytokine comprising a novel p28 molecule and the Epstein-Barr-virus-induced gene 3 (EBI3) molecules. It augments initiation of T helper type 1-mediated immunity by enhancing the proliferation and cytokine production of T cells. In this study, we examined whether a secreted form of IL-27 subunits would inhibit IL-27-mediated immunological responses. COS-7 cells transduced with the mouse (m) p28 gene secreted a monomeric mp28 protein; however, those transduced with the mEBI3 gene did not detect a mEBI3 protein in the culture supernatants. The secreted mp28 prevented the IL-27-mediated signal transduction and activator of transcription 1 phosphorylation and subsequently inhibited the IL-27-mediated intercellular adhesion molecule-1 induction and interferon-gamma production in CD4(+) T cells. We generated mp28-expressing murine carcinoma Colon 26 cells and inoculated a mixture of the mp28- and mIL-27-expressing Colon 26 cells into syngeneic BALB/c mice. Simultaneous production of mp28 and mIL-27 from Colon 26 cells suppressed IL-27-mediated anti-tumour effects in the mice. We examined the p28-mediated immune suppression by inoculating mp28-expressing myoblasts into allogeneic mice. Forced production of mp28 suppressed the allogeneic cytotoxic T-lymphocyte induction and subsequently retarded the graft rejection. Furthermore, production of both mp28 and mp40, which inhibits the functions of IL-12 and IL-23, prolonged the graft survival longer than the grafts expressing either mp28 or mp40. We propose that p28 can be a regulatory subunit for IL-27-mediated cellular immune responses and a possible therapeutic agent to suppress unfavourable immune responses.