Sir, despite the association between Epstein–Barr virus infection and multiple sclerosis being supported by an increasing number of epidemiological, serological and immunological studies (Ascherio and Munger, 2010; Maghzi et al., 2011; Pender, 2011), the mechanisms linking increased immune reactivity to this ubiquitous herpes virus to CNS pathology in patients with multiple sclerosis remain to be demonstrated (Lü nemann and Mü nz, 2009; Giovannoni, 2011). A point of contention is whether an active and persistent Epstein–Barr virus infection in the CNS of patients with multiple sclerosis may sustain a detrimental immu-nopathological response (Pender, 2009; Ascherio and Bar-Or, 2010). In support of this scenario, we have shown high frequen-cies of Epstein–Barr virus latently infected B cells and more spor-adic signs of viral reactivation in post-mortem brain tissue from patients with multiple sclerosis but not patients with other inflam-matory neurological diseases (Serafini et al., 2007, 2010; Aloisi et al., 2010). Presence of Epstein–Barr virus latently infected cells in active multiple sclerosis brain lesions has been confirmed recently (Tzartos et al., 2012). In contrast, other groups did not find Epstein–Barr virus in all or the majority of post-mortem mul-tiple sclerosis brain samples analysed (Hilton et al., 1994; Opsahl and Kennedy, 2007; Willis et al., 2009; Peferoen et al., 2010; Sargsyan et al., 2010; Torkildsen et al., 2010). As recently discussed in Brain (Lassmann et al., 2011), no con-sensus has been reached on technical and interpretation issues relating to the detection of Epstein–Barr virus in the multiple sclerosis brain. Although a variety of techniques [in situ hybrid-ization for Epstein–Barr virus-encoded small RNA (EBER), immuno-histochemistry for Epstein–Barr virus latent and lytic proteins and PCR assays for Epstein–Barr virus nucleic acids] have been used to search for Epstein–Barr virus in multiple sclerosis brain, comparing and interpreting the contrasting results from different studies has been difficult owing to the lack of common procedures and tools. EBER in situ hybridization is considered the most powerful standard method to study the anatomical localization of Epstein– Barr virus–infected cells (Gulley and Tang, 2008). EBERs (EBER1 and EBER2) are small untranslated Epstein–Barr virus transcripts that are abundantly expressed during all phases of Epstein–Barr virus latent infection and generally localize in the cell nucleus. Despite high target specificity and sensitivity of EBER probes, false negative results due to RNA degradation or presence of non-specific staining can invalidate the conclusions of in situ hybridization studies (Gulley and Tang, 2008). Regarding Epstein–Barr virus presence in the multiple sclerosis brain, criticism has been raised toward the high number of EBER-positive cells we found in multiple sclerosis white matter lesions and meninges (par-ticularly inside B cell follicles) (Serafini et al., 2007) and the pres-ence of both nuclear and cytoplasmic EBER signals observed using a peptide nucleic acid (PNA) probe and detection kit from DakoCytomation (Lassmann et al., 2011). The reported absence of EBER signals in brain tissues from cases with other inflammatory neurological diseases or in non-pathological brain and lymphoid tissues (Serafini et al., 2007, 2010) was not deemed sufficient to interpret the EBER signals observed in the multiple sclerosis brain as specific (Lassmann et al., 2011). We reasoned that a sound strategy to confirm or disprove the findings obtained with the commercial EBER in situ hybridization kit (Serafini et al., 2007) was to take advantage of the high sen-sitivity of a radioactive in situ hybridization protocol routinely used by one of the authors (L.M.) at S. Raffaele Hospital in Milan (Muzio et al., 2002, 2005a, b, 2010; Centonze et al., 2009) and the availability of non-commercial EBER probes validated in previous studies (Niedobitek et al., 1991) to analyse EBER doi:10.1093/brain/aws315