Background Female gender, human leukocyte antigen (HLA) DR2, tobacco smoking and Epstein-Barr virus (EBV) are established risk factors for multiple sclerosis (MS). Their possible interaction however, has been sparsely studied. Objectives To investigate possible associations between EBV antibody levels and a range of other recognized MS risk factors. Design, setting and study population Cross-sectional study undertaken in Denmark based on 517 healthy individuals selected from the Danish population. Methods We measured change in mean log (anti-Epstein-Barr viral capsid antigen (VCA) immune globulin G) using linear regression. Results Anti-Epstein-Barr VCA immune globulin G levels were positively correlated with female gender and HLA DR2. Furthermore, current smoking and cumulative tobacco consumption were positively associated with EBV antibody levels. Conclusion The association between Epstein-Barr VCA antibody levels and non-viral MS risk factors support the view that EBV is critically involved in the etiology of MS. These non-viral MS risk factors may be linked with MS risk through EBV-specific immune responses. Multiple Sclerosis 2007; 13: 420-423. http://msj.sagepub.com