M ultiple sclerosis (MS) is characterized by intra-blood-brain barrier immunoglobulin synthesis that persists lifelong. Subcellular fractionation and two-dimensional electrophoresis were used in conjunction with immune precipitation and immunoblotting to identify antigenic determinants for this immunoglobulin. We report that 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein associated with oligodendrocyte/myelin membranes, also present in lymphocytes and retina, is one major target for the humoral response. Antibodies to CNP are detected in sera of 74% of MS patients. The antibodies are IgM and are present in serum in high titer as well as in cerebrospinal fluid. The antibody response is temporally persistent, consistent with systemic immune activation and persistent antigenic stimulation. Moreover, CNP is isolated as an immune complex from MS brain. CNP is expressed as two isoforms, with CNPII identical to CNPI but with a 20-amino acid extension at the amino terminus of CNPII; however, the antibody response is exclusively restricted to CNPI. In contrast, both isoforms bind the C3 complement, providing a plausible mechanism in MS central nervous system (CNS) for opsonization of myelin membrane CNP, mediated via the C3 receptor, and phagocytosis of CNP-Ig immune complexes, mediated by membrane Ig Fc receptors of macrophages and CNS microglia.