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 antibodies 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.