The antigenic specificities of 24 V4‐34‐encoded monoclonal antibodies were compared with the amino acid sequence. The specificities were divided into three categories, red blood cells, B lymphocytes and auto/exoantigens. Six anti‐I monoclonal antibodies, with multiple substitutions in their VH region, did not bind B lymphocytes or auto/exoantigens. Reactivity to these two antigens segregated with the 16 anti‐i monoclonal antibodies, which were derived from the near germline V4‐34 gene. All anti‐i monoclonal antodies bound B lymphocytes, albeit with varying intensities. B‐cell binding correlated with basic amino acids in the VH‐CDR3. Reactivity to auto/exoantigens was demonstrated only by a subset anti‐i monoclonal antibodies and did not correlate with B‐lymphocyte or i‐antigen binding. These anti‐ssDNA reactive monoclonal antibodies had basic amino acids in the VH‐CDR3, strongly supporting the suggested role of arginine in DNA binding. However, an arginine‐rich CDR3 was not enough to ensure DNA reactivity, since six other anti‐i monoclonal antibodies that fulfilled this criteria did not bind ssDNA. Thus it is possible that the anti‐DNA reactivity of V4‐34‐encoded monoclonal antibodies is mediated by the classic antigen‐binding groove generated by the CDRs of the heavy/light chains. In contrast, anti‐B‐cell/i‐antigen reactivity is mediated, unconventionally, by the V4‐34 protein with a dominant influence of the VH‐CDR3.