The multiple nature of heterophile, Paul-Bunnell (P-B) antigen has been suggested by our previous studies as well as those of others. In the present study, two distinct antigens of P-B specificity were defined by means of hemagglutination and immunodiffusion tests in agarose gel; one antigen (BS) was shared by bovine (BRBC) and sheep red blood cells (SRBC) and another antigen (B) was limited to BRBC. Both anti-BS and anti-B antibodies were shown to be present in sera of 106 patients with infectious mononucleosis (IM) whereas they were virtually absent from the sera of the vast majority of patients with diseases other than IM and normal sera. Absorption and agglutination inhibition tests demonstrated the presence of BS and B antigens on horse erythrocytes. Goat erythrocytes were also shown to possess BS antigen, however, B antigen was found on erythrocytes of some but not all individual goats. By means of the previously established procedure, BS antigen was extracted from stromata of BRBC, SRBC and erythrocytes of horse and goat, and B antigen from BRBC and erythrocytes of some goats. BS and B antigens were also extracted from bovine lymphocytes and murine thymus and spleen tissues but not murine erythrocytes.