Lymph node-type T- and B-cell infiltrates with germinal centers are characteristic features of the hyperplastic thymus in early onset myasthenia gravis (EOMG).Epstein-Barr virus (EBV) infection confers survival advantages on B cells, and has recently been implicated in tertiary lymphoid tissue formation in EOMG. We evaluated the frequency of intrathymic EBV-infected B-lineage cells and antiviral immune responses in treatment-naive patients with EOMG. Real-time polymerase chain reaction was performed to quantify the content of genomic EBV DNA (BamHI-W repeat region) in thymic cell suspensions. Serial paraffin sections of EOMG thymi were analyzed for the presence of EBV-encoded RNA by in situ hybridization and for viral gene expression by immunohistochemistry. Humoral and cellular immune responses to viral antigens were quantified by enzyme-linked immunosorbent assay and flow cytometry-based intracellular cytokine staining. We detected minimal levels of viral DNA-corresponding to single viral genomes-in only 6 of 16 hyperplastic EOMG thymi, indicating extreme rarity of viral copy numbers in the investigated thymic samples. That was confirmed by similar rarity of EBV-encoded RNA and viral proteins identified in thymic sections. Furthermore, EBV-specific T- and B-cell responses were unchanged in patients with EOMG. These findings do not support an etiologic role for EBV in the initiation of EOMG.