The heavy chains of the anti-DNA autoantibody D42, derived from an autoimmune NZB/NZW F1 mouse, and the anti-phosphorylcholine (PC) Ab 6G6, obtained from an immunized CBA/J mouse, are both encoded by the same VH11 (S107) germ-line gene. We have combined VH CDR3 exchange, site-directed mutagenesis, transfection of mutant hybridoma cells, and equilibrium affinity measurements of the expressed Abs to analyze the contribution of germ line-encoded genetic elements and somatic mutations to DNA binding affinity as well as to test the possibility that an anti-PC Ab can be converted to an anti-DNA Ab by somatic mutation. Our results indicate that the major contribution to DNA binding affinity is provided by the nonmutated rearranged configuration of the autoantibody and depends on the particular H/L chain pairing and the structure of the VH CDR3 gene segment. Two heavy chain somatic mutations increased the DNA binding affinity of the D42 anti-DNA Ab by about 10-fold. One of these mutations abolished PC binding of the 6G6 Ab, but did not convert it to an anti-DNA. These results suggest that activation of autoreactive B cells bearing germ-line-encoded Ag receptors may be required to initiate the affinity maturation of an anti-DNA response, and that B cells responding by somatic mutation to external Ags, such as PC, may be unable to contribute significantly to the high affinity immune response that is typical of disease-related anti-DNA autoantibodies.