We constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 of the anti-digoxin antibody 26-10 Fab to investigate sequence constraints necessary for high affinity binding in an antibody of known crystal structure. Phage were selected by panning against digoxin and three C-16-substituted analogues. All antigen-positive mutants selected using other analogues also bound digoxin. Among 73 antigen-positive clones, 26 different nucleotide sequences were found. The majority of Fabs had high affinity for digoxin (Ka 3.4 x 10(9) M-1) despite wide sequence diversity. Two mutants displayed affinities 2- and 4-fold higher than the parental antibody. Analysis of the statistical distribution of sequences showed that highest affinity binding occurred with a restricted set of amino acid substitutions at positions H33-35. All clones save two retained the parental Asn-H35, which contacts hapten and hydrogen bonds to other binding site residues in the parental structure. Positions H30-32 display remarkable diversity, with 10-14 different substitutions for each residue, consistent with high affinity binding. Thus complementarity can be retained and even improved despite diversity in the conformation of the N-terminal portion of the H-CDR1 loop.