Elevated cerebrospinal fluid (CSF) IgG and oligoclonal IgG bands on electrophoresis are valuable clinical markers for B cell proliferation in the brains of patients with multiple sclerosis (MS). Using two-dimensional electrophoresis, (2DE) we have established that the humoral immune response in MS brain is characterized by finite clonal complexity for the major Ig classes. An important question is whether this immune response is clonally stable or varies with time, related to the development of new lesions and random entry of B cells into the MS brain. To investigate this, we performed serial electrophoretic studies on CSF obtained from 19 patients with MS; the intervals ranged from 7 to 12 yr, with a mean of 8 yr. These analyses included studies of IgG, IgA, and IgM, and revealed that the humoral immune response in MS is clonally stable over long periods. Spontaneous fluctuations or reduction in CSF IgG levels by drugs did not qualitatively affect B cell clonal proliferation in MS brain, in that dominant bands and spots were not obliterated. It has been asserted that IgG synthesis in MS is nonsense antibody because the spectotypes of IgG isolated from different regions of MS brains differ. Factors other than clonal heterogeneity could account for differences found using one-dimensional analysis. B cell clonal products resolve into unique and well-resolved spots by 2DE; the method is uniquely suitable for analysis of restricted immune responses. Therefore, Ig were isolated from 11 regions of three MS brains and the 2DE patterns were compared. The similarity of the 2DE patterns indicate unequivocally that major clones are distributed uniformly although some clones are more prominent in some brain areas. IgA and IgM isolated from the same areas also showed similar patterns. Furthermore, the patterns of light and heavy chains in brain regions differed from serum but were similar to the autologous CSF, providing new evidence that CSF IgG in MS derives from synthesis in situ. Our results indicate that, once initiated, B cell clonal proliferation persists indefinitely and is little altered qualitatively at a clonal level over time, even when CSF IgG levels change or are altered by drugs. Our results are consistent with allotype and idiotype analysis of Ig production in MS and conflict with nonsense antibody proposals of the origin and nature of in situ synthesized Ig in MS.