Mononuclear cells extracted from regional lymph nodes, blood, spleen and central nervous system of guinea pigs with chronic relapsing experimental allergic encephalomyelitis (r-EAE), adjuvant immunized and untreated controls were cultured for 16 h in microtitre plates, and culture supernatants were then used to measure IgG and IgM, as well as IgG class anti-myelin antibody production by enzyme-linked immunosorbent assays. Increased synthesis of these immunoglobulins and antibodies was found during the course of r-EAE both in intra- and extrathecal compartments. Long-term cultures carried out for 7 days gave similar results but anti-myelin, anti-myelin basic protein and IgG synthesis was most pronounced intrathecally. Agarose isoelectric focusing of supernatants from these cultures showed oligoclonal IgG. These findings indicate in vivo synthesis of autoantibodies within the target for immune attack and a partial sequestration of the immune response to this compartment.