Soluble MHCII (sMHCII) molecules are present in body fluids of healthy individuals and are considered to be involved in the maintenance of self tolerance, and are also related to various diseases. Their concentration increases during in vivo antigen-specific tolerogenic stimulation and it was recently shown that exosome-mediated tolerance is MHCII dependent. At the cellular level, sMHCII proteins compete with membrane MHCII for T-cell receptor binding on CD4+ T cells. Immunoaffinity purification techniques isolated sMHCII antigens from the serum of human serum albumin (HSA) -tolerant mice as a single highly glycosylated protein of ~ 60 000 molecular weight, specifically interacting with anti-class II antibodies in Western blotting and ELISA. Mass spectroscopy showed that these sMHCII proteins were loaded with the tolerogenic peptide as well as multiple self peptides. At the cellular level, sMHCII suppressed antigen-specific, and to a lesser degree antigen-non-specific, spleen cell proliferation and induced CD25 in naive T cells. In T cells activated by antigen-seeded macrophages, sMHCII decreased CD28 and increased CTLA-4 protein expression, while decreasing interleukin-2 and increasing interleukin-10 production. In this case, sMHCII proteins were shown to decrease ZAP-70 and LAT phosphorylation. The results presented here for the first time provide evidence for the role of sMHCII proteins in immune response suppression and maintenance of tolerance, revealing novel regulatory mechanisms for immune system manipulation.