Sera of patients with infectious mononucleosis contain heterophile anti-Paul- Bunnell (PB) antibodies to erythrocytes of numerous mammalian species. Evidence is presented that the corresponding antigen of bovine erythrocytes is not, as previously described, a single molecule, but a series of glycoproteins with glycans terminated with N-glycolylneuraminic acid (Neu5Gc). The latter compound should be an important part of the PB epitope because, in agreement with the results of others, we found that desialylation of the PB antigen abolishes almost completely its activity. We examined three different preparations of GM3 ganglioside for their capacity to bind anti-PB and found that only GM3 from horse erythrocytes containing Neu5Gc exhibited a low although ELISA measurable PB activity. The other two GM3 preparations, from bovine milk and dog erythrocytes, containing N-acetylneuraminic acid (Neu5Ac) bound little if any anti-PB antibodies. This finding confirms a previous report that human erythrocyte Neu5Ac containing sialoglycoprotein with similar O-linked glycans as the PB-antigen of bovine erythrocytes exhibits only very low PB activity (Patarca & Fletcher, 1995, Crit Rev Oncogen., 6: 305). In conclusion, we present a hypothesis that anti-PB antibodies in patients with infectious mononucleosis are formed against infection-induced cell membrane glycoconjugates containing highly immunogenic Neu5Gc.