The 1F6 hybridoma protein, exhibiting the predominant cross-reactive idiotype (CRI) associated with the immune response to p-azophenylarsonate in A/J mice but failing to bind the hapten arsonate, was elicited following immunization with rat anti-CRI [Wysocki, L. J., & Sato, V. (1981) Eur. J. Immunol. 11, 832-839]. The dissociation of idiotype and antigen binding in this hybridoma provides an opportunity to determine structural features involved in antigen binding and idiotypic sites. The complete heavy-chain variable region (VH) amino acid sequence was obtained by automated Edman degradation of the intact chain and fragments due to CNBr cleavage, trypsin digestion, mild acid hydrolysis, and carboxypeptidase A digestion of a CNBr fragment. Comparison of the CRI+ arsonate-nonbinding 1F6 sequence with the CRI+ germ-line VH gene sequence reveals that the 1F6 heavy chain differs from the germ-hne-encoded amino acid sequence at seven positions within VH [Siekevitz, M., Gefter, M. L., Brodeur, P., Riblet, R., & Marshak-Rothstein, A. (1982) Eur. J. Immunol. 12, 1023-1032], The 1F6 VH appears to arise from the CRI+ germ-line VH by somatic mutation at at least seven amino acid residues, each of which could be due to a single nucleotide base change. The diversity (D) gene-encoded segment of 1F6 is similar to that of the CRI+ antigen-binding hybridoma 36-65 except for two amino acid substitutions. Further, the idiotype (CRI) is preserved despite use of a JH4 gene segment in 1F6 as compared to JH2 in all CRI+ arsonate-binding hybridomas examined to date. The lack of antigen binding in the 1F6 hybridoma may be correlated with the presence of a JH4-encoded sequence, although alterations in the light-chain variable region, or elsewhere in the heavy-chain variable region, cannot be excluded. © 1984, American Chemical Society. All rights reserved.