Recent epidemiological and immunological studies provide evidence for an association between Epstein-Barr virus infection and multiple sclerosis, suggesting a role of Epstein-Barr virus infection in disease induction and pathogenesis. A key question in this context is whether Epstein-Barr virus-infected B lymphocytes are present within the central nervous system and the lesions of patients with multiple sclerosis. Previous studies on this topic provided highly controversial results, showing Epstein-Barr virus reactivity in B cells in the vast majority of multiple sclerosis cases and lesions, or only exceptional Epstein-Barr virus-positive B cells in rare cases. In an attempt to explain the reasons for these divergent results, a workshop was organized under the umbrella of the European Union FP6 NeuroproMiSe project, the outcome of which is presented here. This report summarizes the current knowledge of Epstein-Barr virus biology and shows that Epstein-Barr virus infection is highly complex. There are still major controversies, how to unequivocally identify Epstein-Barr virus infection in pathological tissues, particularly in situations other than Epstein-Barr virus-driven lymphomas or acute Epstein-Barr virus infections. It further highlights that unequivocal proof of Epstein-Barr virus infection in multiple sclerosis lesions is still lacking, due to issues related to the sensitivity and specificity of the detection methods.