The CD19+ B‐lymphocyte and CD138+ plasma cell repertoires in cerebrospinal fluid from four patients with monosymptomatic optic neuritis (ON) were analyzed by single‐cell reverse transcriptase polymerase chain reaction. Amplified heavy (H)– and light (L)–chain antibody segments were sequenced and used to identify the rearranged germline and J segment of closest homology. Both the B‐cell and plasma cell repertoires from ON cerebrospinal fluid demonstrated significant clonal expansion. Up to 75% of the amplified H‐ and L‐chain sequences were contained in overrepresented populations and were somatically mutated, consistent with an antigen‐targeted response. The relationship between clonal populations within the CD19+ B lymphocyte and CD138+ plasma cell populations suggests ongoing mutational pressure to refine antigen binding. Our observations demonstrate that an antigen‐driven clonal B‐lymphocyte and plasma cell response is prominent in the initial stages of central nervous system demyelination and suggest that detection of the disease‐relevant antigens in ON may bear on the inciting antigens in chronic inflammatory disorders such as multiple sclerosis. Ann Neurol 2004;56:97–107