Thin-layer polyacrylamide gel isoelectric focusing (PAG IEF), a very high capacity method for separating immunoglobulins (Ig), was performed on cerebrospinal fluid (CSF) and serum. It was followed by antigen immunofixation with measles, mumps, herpes simplex (HSV), and rubella virus antigens and anti-human Ig autoradiography in order to demonstrate viral antibodies in separated Ig zones. Two of 11 control patients and 21 of 25 patients with multiple sclerosis (MS) displayed one or more zones of viral antibodies in the CSF without any counterpart, or with distinctly fainter zones, in the serum. Such reaction patterns were taken to indicate the possibility of intrathecal antibody synthesis. Antibody synthesis to measles was found in one to five zones in 76% of the patients with MS; antibody zones were found to HSV in 36% of the patients, to mumps in 12%, and to rubella in 12%. In 36% of the patients, two or three different antibody specificities (of which one was always measles) were found simultaneously in individual autoradiogram zones. For all viral antibodies detected in the CSF autoradiograms, their counterparts in oligoclonal or polyclonal IgG zones (or both) were demonstrable by PAG IEF of the corresponding CSF. The majority of patients with MS also had one or more oligoclonal CSF IgG zones without known antibody specificity. Antigen immunofixation and autoradiography are mainly qualitative. It is not known whether the viral antibodies present in oligoclonal or polyclonal IgG zones in MS CSF reflect a polyclonal B cell activation, a disease-specific immune reaction, or both.