Abstract To determine the chemical basis of heavy chain allotypic specificity, amino acid analyses were carried out on the heavy chains isolated from the non-specific G-immunoglobulins of a1, a 2 and a3 rabbits. Multiple but consistent differences in composition were found to be associated with each heavy chain allotype. The a2 chains differedf from the a1 chains by an average of 12 residues involving six amino acids, aspartic acid, threonine, serine, proline, alanine, and tyrosine, while the a3 chains differed from the a1 chains by an average of 12 residues involving eight amino acids, arginine, aspartic acid, threonine, proline, alanine, valine, isoleucine, and phenylalanine. The observed differences were not related to antibody content since the same differences were obtained when a comparison was made among the compositions of a1, a2 and a3 chains isolated either from anti-phenylarsonic acid antibody or from anti-phenyltrimethylammonium antibody. Conversely, the few but consistent differences in composition previously seen between the a1 chains of anti-phenylarsonic acid antibody and anti-phenyltrimethylammonium antibody were found to be independent of allotype. Whether the two antibody populations were isolated from an individual animal of a1, a2, or a3 allotype, the anti-phenyltrimethylammonium heavy chain contained one more aspartic acid and one more leucine residue than the anti-phenylarsonic acid heavy chain. From the present data, these amino acid differences appear to be related to antibody specificity rather than to heavy chain subclasses.