AbstractThe mouse hybridoma cell line 40–150 scretes antibodies with high affinity towards the cardiac glycosides digoxin and digitoxin. A spontaneous mutant, 40–150 A2.4, produces and antibody which carries a single residue mutation, Ser → Arg, in its heavy chain (H94) and has an altered specificity. A second order mutant 40–150 A2.4 P.10, produces two antibody molecules, one the same as 40–150 A2.4, the other lacking two residues at the N‐terminus of its H chain, and having a specificity profile approaching that of 40–150 antibody.1The N‐terminus and the position H94 are distant from the antigen‐binding site of the antibody; thus, the structural basic of the specificity changes was not immediately clear. Approximate structures of the 40–150 antibody and its mutants were constructed in the computer, based on atomic coordinates of the homologous mouse antibody McPC 603. Using the program OCNGEN, the torsional space of the polypeptide backbone and side chains around position H94 was uniformly sampled, and the lowest energy conformations were analyzed in detail. The results indicate that when Arg‐H94 is substituted for Ser. Agr‐H94 can hydrogen bond to side chains of Asp‐H101, Arg‐L46, and Asp‐L55. The results in a change in the surface of the combining site which may account for the affinity changes. Deletion of the two N‐terminal residues increases solvent accessibility of Arg‐H94. The solvation may cause a hydrogen bond between Arg‐H94 and Asp‐H101 to be lost, restoring the struc