S107, a phosphocholine-binding myeloma protein, has been cloned in soft agar, and an antigen-binding variant has been isolated and characterized. The variant does not bind phosphocholine attached to carrier or as free hapten in solution but does retain antigenic determinants (idiotypes) of the parent. Chain recombination experiments suggest that the defect in binding is entirely in the heavy chain. Amino acid sequence analysis showed a single substitution--glutamic acid to alanine at position 35--in the first hypervariable or complementarity-determining region. In terms of the three-dimensional model of the phosphocholine-binding site, glutamic acid-35 provides a hydrogen bond to tyrosine-94 of the light chain that appears to be critical for stability of this portion of the binding site. The removal of this bond and the presence of the smaller alanine side chain is thus consistent with the loss in binding activity. These results suggest that small numbers of substitutions in antibodies, such as those presumably introduced by somatic mutation, may in some situations be effective in altering antigen-binding specificity.