A well-defined antigen/antibody system was used to evaluate the effect of immune tolerance on the spectrum of specificities of natural antibodies. The antibody used in this study, anti-Gal, is a naturally occurring, polyclonal IgG that constitutes 1% of the circulating IgG in humans. We have previously shown that anti-Gal, purified from AB sera, specifically interacts with glycosphingolipids bearing a Gal alpha 1----3Gal epitope, but not with the closely related B antigen in which the penultimate galactose of the Gal alpha 1----3Gal epitope is fucosylated Gal alpha 1----3(Fuc alpha 1----2)Gal. This narrow specificity was assumed to be the result of an effective immune tolerance mechanism that prevents the expression of antibody clones that can recognize both the Gal alpha 1----3Gal and the self B epitopes. If the assumption that immune tolerance determines the range of anti-Gal specificity is correct, then anti-Gal from individuals lacking the B antigen (A and O blood types) would be expected to interact with both Gal alpha 1----3Gal and Gal alpha 1----3(Fuc alpha 1----2)Gal epitopes. In this study, anti-Gal from the serum of individuals of various blood types was purified by affinity chromatography on Gal alpha 1----3Gal adsorbent and tested for its reaction with the B antigen. Whereas anti-Gal from AB and B individuals only reacted with Gal alpha 1----3Gal epitopes, anti-Gal from A and O individuals reacted with both Gal alpha 1----3Gal and B epitopes. Furthermore, it was determined that the majority of anti-B reactivity in A and O individuals is in fact anti-Gal antibodies capable of recognizing both Gal alpha 1----3Gal and B epitopes. It can be concluded from these results that immune tolerance accurately controls the spectrum of natural antibody specificities by preventing the production of antibody clones that can interact with self antigens.