Five murine A/J strain anti-digoxin mAb (35-20, 40-40, 40-120, 40-140, and 40-160) have highly homologous H and L chain V regions, only differing by somatic mutation, yet differ in affinity and specificity. The availability of the VH and VL genomic clones from one hybridoma, 40-140, has now allowed studies involving in vitro mutagenesis and chain recombination among these five hybridomas. To determine the relative contributions of the mutations found in either VH or VL to the overall binding properties of these antibodies, we recombined the 40-140VH with the VL of each hybridoma. The 40-140VH gene was transfected into hybridoma variants that produce only VL. The recombinant antibodies show that the mutations present in VH, rather than in VL, affect the fine specificity properties of these antibodies, whereas, the mutations among both VH and VL chains are important in determining antigen affinity. From mutations present in VH that affect fine specificity properties, the comparison of the antibody sequences, and from the previously measured binding properties, we predicted and tested selected VH mutations for their ability to alter specificity or affinity by doing site-directed in vitro mutagenesis. The results for the somatic mutations found in this group of antibodies show: 1) VH mutations control the fine specificity properties that distinguish different members of this group; 2) in particular, VH residues 54 and 55 in CDR2 control the distinguishing characteristics of specificities between these antibodies; and 3) by mutagenesis, we had the unusual result of being able to alter Ag specificity without affecting affinity. A computer model of the 40-140 antibody binding site was generated which indicates that VH residues 54 and 55 are highly accessible.