The presence of a tolerogen in mouse serum within 1 hr of antigen feeding prompted further study. Therefore, serum from mice fed with ovalbumin (OVA) was subjected to various immunochemical and biological tests. The appearance of tolerogen in serum was concomitant with the presence of OVA detected by a specific ELISA. Absorption of tolerogenic serum with anti-OVA antibody coupled to Sepharose beads effectively removed the tolerogenic moiety from the serum and confirmed that not only was tolerogenicity associated with the presence of antigen, but that binding sites for antibody were intact on this tolerogenic form of OVA. Finally, serum fractions from antigen-fed mice were assayed for total protein content, ELISA-detectable OVA and in vivo effect on systemic immunity. The only serum fraction in which immunoreactive OVA was detected contained proteins close to the molecular weight of native OVA and induced significant immune suppression in recipients. Serum fractions lacking immunoreactive OVA were not significantly tolerogenic in vivo. These experiments confirm that when OVA is absorbed across the gut mucosa it is subtly altered into a tolerogenic form. The recognition of gut-processed OVA by T-suppressor cells is discussed.