We report a case of herpetic brainstem encephalitis (HBE) retrospectively diagnosed in adult patient. Conventional immunovirological studies failed to disclose the etiology of this patient's affection. An isoelectric focusing-antigen overlay (IEF-O) technique showed that the target of one of the four cerebrospinal fluid oligoclonal bands was herpes simplex virus (HSV)-1 glycoprotein B, indicating a specific anti-HSV immunoresponse restricted to the CNS. IEF-O may represent a useful support for in vivo diagnosis of HBE.