We previously reported that human autoantibodies with cold agglutinin activity contained a single human VH gene segment (VH4-21) which was also responsible for the cross-idiotypic specificity characteristic of the cold agglutinin response. To confirm and extend this observation we have analyzed at the nucleotide level the H and L chains of six new cold agglutinin molecules derived from different patients. We found that regardless of whether the antibody recognizes the i or the I red cell Ag, restriction at the VH gene segment level is absolute. We also found that even in the absence of somatic mutation the VH4-21 gene segment can encode both anti-i and anti-I specificities. Finally, although the VH4-21 gene segment is essential for cold agglutinin activity, the other genetic elements that contribute to the V region of the antibody molecules can be extremely diverse. The structural information provided in this report sharply restricts the requirement for encoding pathogenic cold agglutinin activity to one of the components of the H chain V region, specifically the VH gene segment. The implications of this apparently absolute requirement for a single VH gene segment, unprecedented in the human autoimmune response, are discussed.