The heavy-chain CDR3 region of the high affinity (K(a) = 1.3 x 10(10) M(-)1) anti-digoxin monoclonal antibody 26-10 was modified previously to shift its specificity, by substitution of tryptophan 100 by arginine, toward binding analogs of digoxin containing substitutions at position 16. To further change specificity, two 5-mer libraries of the randomly mutagenized phage-displayed 26-10 HCDR3 region (positions 94-98) were panned against digoxin-bovine serum albumin (BSA) as well as against 16-acetylgitoxin-BSA. When a mutant Fab that binds 16-substituted analogs preferentially was used as a parent sequence, clones were obtained with affinities for digoxin increased 2-4-fold, by panning on digoxin-BSA yet retaining the specificity shift. Selection on 16-acetylgitoxin-BSA, however, resulted in nine clones that bound gitoxin (16-OH) up to 150-fold higher than the wild-type 26-10, due to a consensus mutation of Ser(H95) to Gly(H95). The residues at both position H95 (serine) and position H100 (tryptophan) contact hapten in the crystal structure of the Fab 26-10-digoxin complex. Thus, by mutating hapten contact residues, it is possible to reorder the combining site of a high affinity antibody, resulting in altered specificity, yet retain or substantially increase the relative affinity for the cross-reactive ligand.