Presence of oligoclonal bands (OCB) in cerebrospinal fluid (CSF) is a diagnostic hallmark of multiple sclerosis (MS). However, up to 10% of patients were OCB negative in routine laboratory tests. The aim of this study was to determine whether there is at least an oligoclonal restriction of intrathecal antibody synthesis against measles, rubella and/or varicella zoster virus (MRZ-specific OCB) in CSF from oligoclonal bands-negative patients with MS.CSF and serum samples from 17 well-defined OCB-negative patients with MS were analysed for MRZ-specific OCB. We performed isoelectric focusing (IEF) combined with affinity blotting using viral antigens, detection with a highly sensitive chemiluminescence technique and recording with X-ray films. Controls included 18 OCB-positive patients with MS and 11 patients with pseudotumor cerebri (PTC).Exclusive or predominant MRZ-specific OCB in CSF against at least one virus species were present in 8 of 17 patients with MS (47.1%; P = 0.0422), suggesting an oligoclonal intrathecal immune response, although OCB of total IgG were absent. Only a very weak oligoclonal reaction against varicella zoster virus in CSF from one of the PTC controls was detectable. Thirteen of 18 (72.2%; P = 0.0013) OCB-positive patients with MS showed also MRZ-specific oligoclonal bands against at least 1 neurotropic virus in CSF.MRZ-specific OCB argue for existence of a chronic intrathecal immune reaction also in routine laboratory-OCB-negative patients with MS. This phenomenon reflects oligoclonal restriction of the humoral immunoreaction as well as polyspecific intrathecal antibody synthesis, which are both characteristics in the chronic inflammatory process of MS.