The objective of this study was to evaluate the association between infectious mononucleosis (IM) and multiple sclerosis (MS). In a historical prospective study we used records from the Danish State Serum Institute in which heterophile antibody tests (HA) for infectious mononucleosis were performed for all Danish patients for a number of years. Included in the analysis were 6853 HA-positive people and 12,886 HA-negative people. A search for the presence of these people in the nationwide Danish Multiple Sclerosis Registry (DMSR) was performed. Among the HA-positive people 16 cases of MS were found. The expected number for a matched Danish population was 5.70, the risk ratio being 2.81. No cases had developed MS before contracting infectious mononucleosis. Among the HA-negative people the expected number of MS patients were found. Although Epstein-Barr virus is not suggested in itself to be the cause of multiple sclerosis, a hypothesis is put forward that it is a cofactor in the pathogenesis of this disease.