The immune response to p-azophenyl arsonate (Ars) in A/J mice is dominated by a cross-reactive idiotype (CRI or IdCR). IdCR+ hybridoma proteins 1F6 and 3D 10 produced in a single mouse by immunization with a monoclonal anti-IdCR antibody did not bind Ars [Wysocki, L., & Sato, V. (1981) Eur. J. Immunol. 11, 832–839]. The preservation of idiotype coupled with lack of antigen binding in the same molecules provoked an examination of their primary structures in order to localize sites involved in binding to antigen and to anti-idiotypes. The VHsequence of antibody 3D 10 was determined by Edman degradation of intact chains and fragments generated by CNBr, hydroxylamine, and o-iodosobenzoic acid cleavage, by trypsin and V8 protease digestion, and by sequence analysis of mRNA. The 1F6 VHsequence was reported previously [Smith, J. A., & Margolies, M. N. (1984) Biochemistry 23, 4726–4732]. The VL sequences of 1F6 and 3D 10 were determined by Edman degradation of intact chains and peptides generated by cleavage with o-iodosobenzoic acid and digestion with trypsin and chymotrypsin. Both 1F6 and 3D 10 are encoded by the same VH, Vk, D, and Jk gene segments as are IdCR+ Ars-binding antibodies. However, 1F6 and 3D10 employ the JH4 gene segment rather than JH2. Antibodies 1F6 and 3D 10 share several somatic mutations, suggesting a common clonal origin, but manifest individual mutations as well. By comparison with Ars-binding IdCR+ molecules, the substitutions in 1F6 and 3D 10 likely responsible for the lack of Ars binding are localized to the heavy chain D-JH junction and/or to a substitution in light chain CDR 3. © 1987, American Chemical Society. All rights reserved.